Compositions and methods for the therapy and diagnosis of inflammatory bowel disease

ABSTRACT

Compositions and methods for the therapy and diagnosis of Inflammatory Bowel Disease (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, are disclosed. Illustrative compositions comprise one or more bacterial polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of IBD.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of PCT Application No.PCT/US02/40422, filed on Dec. 16, 2002, which is related to U.S.Provisional Patent Application Nos. 60/426,835, filed Nov. 15, 2002,60/396,242, filed Jul. 16, 2002, and 60/341,830, filed Dec. 17, 2001,which are all incorporated herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to therapy and diagnosis ofCrohn's Disease and Ulcerative Colitis (collectively referred to asInflammatory Bowel Disease, or IBD). The invention is more particularlyrelated to polypeptides comprising at least a portion of a protein thatis recognized, and to which individuals with IBD mount an aberrantimmune response, and to polynucleotides encoding such polypeptides. Suchpolypeptides and polynucleotides are useful in pharmaceuticalcompositions, e.g., vaccines, and other compositions for the diagnosisand treatment of IBD.

2. Description of the Related Art

Crohn's Disease and Ulcerative Colitis (collectively referred to asInflammatory Bowel Disease or IBD) are chronic, inflammatory diseases ofthe gastrointestinal tract. While the clinical features vary somewhatbetween these two disorders, both are characterized by abdominal pain,diarrhea (often bloody), a variable group of ‘extra-intestinal’manifestations (such as arthritis, uveitis, skin changes, etc) and theaccumulation of inflammatory cells within the small intestine and colon(observed in pathologic biopsy or surgical specimens).

IBD affects both children and adults, and has a bimodal age distribution(one peak around 20, and a second around 40). IBD is a chronic, lifelongdisease, and is often grouped with other so-called “autoimmune”disorders (e.g. rheumatoid arthritis, type I diabetes mellitus, multiplesclerosis, etc). IBD is found almost exclusively in the industrializedworld. The most recent data from the Mayo Clinic suggest an overallincidence greater than 1 in 100,000 people in the United States, withprevalence data in some studies greater than 1 in 1000. There is a cleartrend towards the increasing incidence of IBD in the US and Europe,particularly Crohn's Disease. The basis for this increase is notpresently clear. As such, IBD represents the 2^(nd) most commonautoimmune disease in the United States (after rheumatoid arthritis).

Treatment of IBD is varied. First line therapy typically includessalicylate derivatives (e.g. 5-ASA) given orally or rectally. Responserates in uncomplicated Crohn's Disease are approximately 40% (comparedto 20% for placebo). Corticosteroids remain a mainstay in the treatmentof patients with more “refractory” disease, despite the untowardside-effects. Newer treatment options include anti-metabolites (e.g.methotrexate, 6-mercaptopurine) and immunomodulators (e.g. Remicade—achimeric human antibody directed at the TNFα receptor).

In spite of considerable research into therapies for these disorders,IBD remains difficult to diagnose and treat effectively. Furthermore,there are no clear laboratory tests that are diagnostic for IBD, nor arethere suitable laboratory tests that serve as “surrogate marker” thatare uniformly useful to follow the course of disease in patients.Accordingly, there is a need in the art for improved methods fordetecting and treating such inflammatory bowel diseases. The presentinvention fulfills these needs and further provides other relatedadvantages.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of flagellin clones with percent similarity torelated flagellin B from the rumen anaerobe, Butyrivibrio fibrisolvens.

FIG. 2 a shows Western Blot analysis of serum antibody response torecombinant flagellins cBir-1 and Fla^(X) and fragments.

FIG. 2 b shows titration of serum anti-flagellin antibody againstrecombinant flagellins cBir-1 and Fla^(X).

FIG. 2 c shows the correlation of colitis score with serum anti-Fla^(X).

FIG. 2 d shows association of anti-flagellin antibody with humaninflammatory bowel diseases. NL is negative population, UC is ulcerativecolitis positive, IC is inflammatory control, CD is Crohn's Diseasepositive and UC+ and CD+ pANCA+ are ulcerative colitis pANCA positiveand Crohn's Disease pANCA positive.

FIGS. 3 a and b show cytokine release by donors stimulated withflagellin.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides polynucleotidecompositions comprising a sequence selected from the group consistingof:

-   -   (a) sequences provided in SEQ ID NOs: 75, 83, 85, 1-37, 51-74,        76-78 and 87;    -   (b) complements of the sequences provided in SEQ ID NOs: 75, 83,        85, 1-37, 51-74, 76-78 and 87;    -   (c) sequences consisting of at least 20, 25, 30, 35, 40, 45, 50,        75 and 100 contiguous residues of a sequence provided in SEQ ID        NOs: 75, 83, 85, 1-37, 51-74, 76-78 and 87;    -   (d) sequences that hybridize to a sequence provided in SEQ ID        NOs: 75, 83, 85, 1-37, 51-74, and 76-78, under moderate or        highly stringent conditions;    -   (e) sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%,        98% or 99% identity to a sequence of SEQ ID NOs: 75, 83, 85,        1-37, 51-74, 76-78 and 87;    -   (f) degenerate variants of a sequence provided in SEQ ID NOs:        75, 83, 85, 1-37, 51-74, 76-78 and 87.

The present invention, in another aspect, provides polypeptidecompositions comprising an amino acid sequence that is encoded by apolynucleotide sequence described above.

The present invention further provides polypeptide compositionscomprising an amino acid sequence selected from the group consisting ofsequences recited in SEQ ID NOs: 79, 84, 86, 80-82, 38-50 and 88-89.

In certain preferred embodiments, the polypeptides and/orpolynucleotides of the present invention are immunogenic, i.e., they arecapable of eliciting an immune response, particularly a humoral and/orcellular immune response, as further described herein.

The present invention further provides fragments, variants and/orderivatives of the disclosed polypeptide and/or polynucleotidesequences, wherein the fragments, variants and/or derivatives preferablyhave a level of immunogenic activity of at least about 50%, preferablyat least about 70% and more preferably at least about 90% of the levelof immunogenic activity of a polypeptide sequence set forth in SEQ IDNOs: 79, 84, 86, 80-82, and 38-50 or a polypeptide sequence encoded by apolynucleotide sequence set forth in SEQ ID NOs: 75, 83, 85, 1-37,51-74, 76-78 and 88-89.

The present invention further provides polynucleotides that encode apolypeptide described above, expression vectors comprising suchpolynucleotides and host cells transformed or transfected with suchexpression vectors.

Another aspect of the present invention provides for isolatedantibodies, or antigen-binding fragment thereof, that specifically bindto the polypeptides of the present invention. In one embodiment of theinvention, the antibody may be a monoclonal antibody. In a furtherembodiment the antibody is a human antibody or an antibody that has beenhumanized. In yet further embodiments, the antibodies of the presentinvention bind to flagellin proteins and in one embodiment theantibodies are neutralizing antibodies against flagellin proteins. In anadditional embodiment, said antibodies block the interaction between aflagellin protein and a Toll-like receptor. In one particularembodiment, the Toll-like receptor is TLR5.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusions proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitating theexpression, purification and/or immunogenicity of the polypeptide(s).

The present invention also provides, in other aspects, oligonucleotidesthat hybridize to the polynucleotides of the present invention. In oneembodiment, the oligonucleotides hybridize to the polynucleotides of thepresent invention under highly stringent conditions. In one embodiment,the oligonucleotides hybridize to polynucleotides that encode flagellinproteins.

The present invention further provides, in one aspect, methods ofstimulating and/or expanding T cells specific for an enteric bacterialprotein, comprising contacting T cells with at least one componentincluding but not limited to, polypeptides or polynucleotides of thepresent invention, antigen-presenting cells that express apolynucleotide of the present invention under conditions and for a timesufficient to permit the stimulation and/or expansion of T cells. In oneembodiment of the invention the T cells are CD4+ T cells. In a furtherembodiment, the CD4+ T cells mediate a decrease in inflammation in thecolon. In another embodiment the T cells are specific for a flagellinpolypeptide.

The present invention, in one aspect, also provides for populations of Tcells produced according to the methods described herein. In oneembodiment, said T cells produce cytokines that may include, but are notlimited to, interleukin 10 (IL-10), interferon-β (IFN-β), interleukin 4(IL-4), interleukin 12 (IL-12), transforming growth factor beta (TGF

or interleukin 18 (IL-18). In preferred embodiments, the T cells produceIL-10 and/or TGFβ.

Within other aspects, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide as describedabove and a physiologically acceptable carrier.

Within a related aspect of the present invention, the pharmaceuticalcompositions, e.g., vaccine compositions, are provided for prophylacticor therapeutic applications. Such compositions generally comprise animmunogenic polypeptide or polynucleotide of the invention and animmunostimulant, such as an adjuvant.

The present invention further provides pharmaceutical compositions thatcomprise: (a) an antibody or antigen-binding fragment thereof thatspecifically binds to a polypeptide of the present invention, or afragment thereof; and (b) a physiologically acceptable carrier.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusions proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitating theexpression, purification and/or immunogenicity of the polypeptide(s).

Within further aspects, the present invention provides pharmaceuticalcompositions comprising: (a) T cells specific for a polypeptide asdescribed above and (b) a pharmaceutically acceptable carrier orexcipient. Illustrative T cells include T cells expressing a variety ofcytokines including interleukin 10 (IL-10), interferon-β (IFN-β),interleukin 4 (IL-4), interleukin 12 (IL-12), transforming growth factorbeta (TGF

or interleukin 18 (IL-18). In preferred embodiments, the T cells produceIL-10 and/or TGFβ.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) a pharmaceutically acceptablecarrier or excipient. Illustrative antigen presenting cells includedendritic cells, macrophages, monocytes, fibroblasts and B cells.

Within related aspects, pharmaceutical compositions are provided thatcomprise: (a) T cells specific for a polypeptide as described above oran antigen presenting cell that expresses a polypeptide as describedabove and (b) an immunostimulant. Illustrative immunostimulants includeadjuvants such as Freund's Incomplete Adjuvant; Freund's CompleteAdjuvant; Merck Adjuvant 65; AS-1, AS-2; aluminum hydroxide gel;aluminum phosphate; a salt of calcium, iron or zinc; an insolublesuspension of acylated tyrosine acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A, QS21, aminoalkyl glucosaminide4-phosphates, or quil A.

In a related aspect, the present invention provides a method ofstimulating an immune response in a mammal, comprising administering tothe mammal the compositions described above. In one embodiment, theimmune response comprises T cells that produce a cytokine including, butnot limited to, interleukin 10 (IL-10), interferon-β (IFN-β),interleukin 4 (IL-4), interleukin 12 (IL-12), transforming growth factorbeta (TGF

or interleukin 18 (IL-18). Particularly illustrative cytokines compriseIL-10 and/or TGFβ.

In a related aspect, the present invention provides a method ofdecreasing gastrointestinal inflammation associated with inflammatorybowel disease in a mammal, comprising administering to said mammal thecompositions of the present invention.

Another aspect of the present invention provides for a method ofdetecting the presence of inflammatory bowel disease in a mammalcomprising contacting a biological sample from the mammal, wherein saidbiological sample comprises antibodies, with the polypeptides describedabove, detecting in the sample an amount of antibody that binds to thepolypeptide; and comparing the amount of bound antibody to apredetermined cut-off value and therefrom determining the presence ofinflammatory bowel disease in the mammal. Illustrative biologicalsamples include sera, stool, tissue or other material obtained bycolonoscopy, ileoscopy, esophagogastroduodenoscopy (EGP), or surgery. Inone particular embodiment the polypeptide comprises a flagellinpolypeptide.

Another aspect of the present invention provides for a method ofdetecting the presence of inflammatory bowel disease in a mammalcomprising contacting a biological sample from the mammal, wherein saidbiological sample comprises polynucleotides, with at least oneoligonucleotide that is at least in part complementary to apolynucleotide described above, detecting in the sample an amount of apolynucleotide that hybridizes to said oligonucleotide; and comparingthe amount of said polynucleotide that hybridizes to saidoligonucleotide to a predetermined cut-off value, and therefromdetermining the presence of inflammatory bowel disease in a mammal. Inone embodiment, the oligonucleotide hybridizes under moderatelystringent conditions. In a particular embodiment, the polymerase chainreaction is used to determine the amount of polynucleotide thathybridizes to said oligonucleotide. In another embodiment, ahybridization assay is used to determine the amount of polynucleotidethat hybridizes to said oligonucleotide. Illustrative biological samplescomprising polynucleotides include sera, stool, tissue or other materialobtained by colonoscopy or colonic biopsy, ileoscopy,esophagogastroduodenoscopy (EGP), or surgery. In one particularembodiment, the polynucleotide encodes a flagellin protein.

Another aspect of the present invention provides for a method ofstimulating and/or expanding B cells that produce antibodies specificfor an enteric bacterial protein, comprising contacting B cells with thepolypeptides or polynucleotides mentioned above under conditions and fora time sufficient to permit the stimulation and/or expansion of B cells.In one embodiment the B cells produce antibodies that bind to aflagellin protein. In another embodiment said antibodies areneutralizing antibodies against a flagellin protein. In anotherembodiment, the antibodies block the interaction between a flagellinprotein and a Toll-like receptor. In one particular embodiment, theToll-like receptor is TLR5.

Within related aspects, the present invention provides for populationsof B cells generated as described above.

Within further aspects, the present invention provides a method ofidentifying bacterial antigens associated with inflammatory boweldisease in a mammal, comprising contacting a biological sample thatcomprises T cells with the polynucleotides, or polypeptides describedabove, or antigen-presenting cells that express a polynucleotidedescribed herein, under conditions and for a time sufficient to permitthe stimulation and/or expansion of T cells, and further, detecting inthe sample the magnitude of said stimulation and/or expansion of Tcells; and, comparing the magnitude of said stimulation and/or expansionto a predetermined cut-off value, and therefrom identifying bacterialantigens associated with inflammatory bowel disease in the mammal. Inone embodiment, the mammal is a human. In a further embodiment themammal is a mouse. Illustrative mouse strains are C3H/HeJ Bir, BALB/cIL-10−/−, B6 IL-10−/−, B10 IL-10−/−, MDR1a −/−, TCRα−/−, IL-2−/−, IL-2R−/−, mice with DSS (Dextransodiumsulfate) induced colitis, Gα_(ai)−/−,and CD45 RB transgenic mice. In one particular embodiment, the strain ofmouse is C3H/HeJ Bir. In another embodiment, the mammal is a rat. In oneparticular embodiment, the rat is an HLA-B27-transgenic rat.

In certain other aspects, the present invention provides methods ofmonitoring the progression of inflammatory bowel disease in a mammal,comprising the steps of: (a) obtaining a biological sample from themammal, wherein said biological sample comprises antibodies; (b)contacting the biological sample with a polypeptide described herein;(c) detecting in the sample an amount of antibody that binds to thepolypeptide; and (d) repeating steps (a), (b), and (c) using abiological sample obtained from the mammal at a subsequent point intime; and (e) comparing the amount of bound antibody in step (c) to theamount of bound antibody in step (d) and therefrom monitoring theprogression of inflammatory bowel disease in the mammal.

Another aspect of the present invention provides methods of identifyingbacterial antigens associated with inflammatory bowel disease in a firstmammal, comprising the steps of: (a) obtaining a biological sample fromsaid first mammal wherein said biological sample comprises DNA fromcecal bacteria; (b) constructing an expression library with said DNA;(c) screening said expression library with sera from either said firstmammal or a second mammal with inflammatory bowel disease; therebyidentifying bacterial antigens associated with inflammatory boweldisease. In one embodiment, both the first and second mammals are mice.In a related embodiment, said first mammal is a C3H/HeJ Bir mouse andsaid second mammal is a different strain of mouse including BALB/cIL-10−/−, B6 IL-10−/−, MDR1a −/−, or CD45 RB transgenic mice. In anotherrelated embodiment, said first mammal is a mouse and said second mammalis a human.

In certain other aspects, the present invention provides methods ofmonitoring the progression of inflammatory bowel disease in a mammal,comprising the steps of (a) obtaining a biological sample from saidmammal, wherein said biological sample comprises polynucleotides; (b)contacting said sample with at least one oligonucleotide that is atleast in part complementary to a polynucleotide described herein; (c)detecting in the sample an amount of a polynucleotide that hybridizes tosaid oligonucleotide; (d) repeating steps (a), (b), and (c) using abiological sample obtained from said mammal at a subsequent point intime; and (e) comparing the amount of said polynucleotide thathybridizes to said oligonucleotide in step (c) to the amount of saidpolynucleotide that hybridizes to said oligonucleotide in step (d); andtherefrom monitoring the progression of inflammatory bowel disease in amammal. In one embodiment, the oligonucleotide hybridizes undermoderately stringent conditions. In one particular embodiment, thepolymerase chain reaction is used to determine the amount ofpolynucleotide that hybridizes to said oligonucleotide. In anotherembodiment, the amount of polynucleotide that hybridizes to saidoligonucleotide is determined using a hybridization assay. Illustrativebiological samples are sera, stool, tissue or other material obtained bycolonoscopy or colonic biopsy, ileoscopy, esophagogastroduodenoscopy(EGP), or surgery. In a related embodiment, the polynucleotide comprisesa polynucleotide that encodes a flagellin protein.

Other aspects of the present invention provides diagnostic kitscomprising at least one oligonucleotide as described herein. In relatedaspects, a diagnostic kit may comprise at least one antibody asdescribed herein, and a detection reagent, wherein the detection reagentcomprises a reporter group.

In certain aspects, the present invention provides a diagnostic kitcomprising a portion of at least one or more polypeptides describedherein, wherein said portion can be bound by an antibody; and adetection reagent comprising a reporter group. In a related embodiment,said portion of at least one or more polypeptides is immobilized on asolid support. Illustrative detection reagents comprise ananti-immunoglobulin, protein G, protein A, or a lectin. Illustrativereporter groups comprise radioactive groups, fluorescent groups,luminescent groups, enzymes, biotin, or dyes.

Within yet another aspect, the present invention provides a method foridentifying an inflammatory bowel disease type in a patient, comprising:(a) obtaining an antibody comprising biological sample from a patient;(b) contacting the biological sample with a polypeptide of claim 2; (c)detecting in the sample an amount of antibody that binds to thepolypeptide; and (d) comparing the amount of bound antibody to apredetermined value associated with and therefrom subdividing theinflammatory bowel disease type. Within one embodiment the disease typeis selected from ulcerative colitis and Crohn's Disease. Within anotherembodiment the polypeptide comprises a flagellin polypeptide.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

SEQ ID NO:1 is the determined cDNA sequence for 76779.

SEQ ID NO:2 is the determined cDNA sequence for 76780.

SEQ ID NO:3 is the determined cDNA sequence for 76959.

SEQ ID NO:4 is the determined cDNA sequence for 76960.

SEQ ID NO:5 is the determined cDNA sequence for 76961.

SEQ ID NO:6 is the determined cDNA sequence for 76781.

SEQ ID NO:7 is the determined cDNA sequence for 76962.

SEQ ID NO:8 is the determined cDNA sequence for 76782.

SEQ ID NO:9 is the determined cDNA sequence for 76963.

SEQ ID NO:10 is the determined cDNA sequence for 76964.

SEQ ID NO:11 is the determined cDNA sequence for 77529.

SEQ ID NO:12 is the determined cDNA sequence for 76965.

SEQ ID NO:13 is the determined cDNA sequence for 76966.

SEQ ID NO:14 is the determined cDNA sequence for 76967.

SEQ ID NO:15 is the determined cDNA sequence for 76968.

SEQ ID NO:16 is the determined cDNA sequence for 77530.

SEQ ID NO:17 is the determined cDNA sequence for 76969.

SEQ ID NO:18 is the determined cDNA sequence for 76970.

SEQ ID NO:19 is the determined cDNA sequence for 76971.

SEQ ID NO:20 is the determined cDNA sequence for 77073.

SEQ ID NO:21 is the determined cDNA sequence for 76972.

SEQ ID NO:22 is the determined cDNA sequence for 76973.

SEQ ID NO:23 is the determined cDNA sequence for 76974.

SEQ ID NO:24 is the determined cDNA sequence for 77074.

SEQ ID NO:25 is the determined cDNA sequence for 77531.

SEQ ID NO:26 is the determined cDNA sequence for 76975.

SEQ ID NO:27 is the determined cDNA sequence for 77075.

SEQ ID NO:28 is the determined cDNA sequence for 76976.

SEQ ID NO:29 is the determined cDNA sequence for 76977.

SEQ ID NO:30 is the determined cDNA sequence for 77532.

SEQ ID NO:31 is the determined cDNA sequence for 77533.

SEQ ID NO:32 is the determined cDNA sequence for 77534.

SEQ ID NO:33 is the determined cDNA sequence for 77535.

SEQ ID NO:34 is the determined cDNA sequence for 77076.

SEQ ID NO:35 is the determined cDNA sequence for 77536.

SEQ ID NO:36 is the determined cDNA sequence for 77538.

SEQ ID NO:37 is the determined cDNA sequence for 77539.

SEQ ID NO:38 is the amino acid sequence encoded by 76779.

SEQ ID NO:39 is the amino acid sequence encoded by 76780.

SEQ ID NO:40 is the amino acid sequence encoded by 76959.

SEQ ID NO:41 is the amino acid sequence encoded by 76959.

SEQ ID NO:42 is the amino acid sequence encoded by 76781.

SEQ ID NO:43 is the amino acid sequence encoded by 76782.

SEQ ID NO:44 is the amino acid sequence encoded by 76967.

SEQ ID NO:45 is the amino acid sequence encoded by 76969.

SEQ ID NO:46 is the amino acid sequence encoded by 76972.

SEQ ID NO:47 is the amino acid sequence encoded by 76974.

SEQ ID NO:48 is the amino acid sequence encoded by 76975.

SEQ ID NO:49 is the amino acid sequence encoded by 76977.

SEQ ID NO:50 is the amino acid sequence encoded by 77076.

SEQ ID NO:51 is the determined cDNA sequence for 73261.

SEQ ID NO:52 is the determined cDNA sequence for 73262.

SEQ ID NO:53 is the determined cDNA sequence for 73263.

SEQ ID NO:54 is the determined cDNA sequence for 73264.

SEQ ID NO:55 is the determined cDNA sequence for 73266.

SEQ ID NO:56 is the determined cDNA sequence for 73267.

SEQ ID NO:57 is the determined cDNA sequence for 73268.

SEQ ID NO:58 is the determined cDNA sequence for 73269.

SEQ ID NO:59 is the determined cDNA sequence for 73270.

SEQ ID NO:60 is the determined cDNA sequence for 73272.

SEQ ID NO:61 is the determined cDNA sequence for 73273.

SEQ ID NO:62 is the determined cDNA sequence for 73274.

SEQ ID NO:63 is the determined cDNA sequence for 73275.

SEQ ID NO:64 is the determined cDNA sequence for 73037.

SEQ ID NO:65 is the determined cDNA sequence for 75038.

SEQ ID NO:66 is the determined cDNA sequence for 75039.

SEQ ID NO:67 is the determined cDNA sequence for 75040.

SEQ ID NO:68 is the determined cDNA sequence for 75041.

SEQ ID NO:69 is the determined cDNA sequence for 75042.

SEQ ID NO:70 is the determined cDNA sequence for 75044.

SEQ ID NO:71 is the determined cDNA sequence for 75045.

SEQ ID NO:72 is the determined cDNA sequence for 75046.

SEQ ID NO:73 is the determined cDNA sequence for 75047.

SEQ ID NO:74 is the determined cDNA sequence for 75048.

SEQ ID NO:75 is the full-length determined cDNA sequence for 83537, alsoreferred to as Flagellin X.

SEQ ID NO:76 is the determined cDNA sequence for the amino terminalconserved end of Flagellin X.

SEQ ID NO:77 is the determined cDNA sequence for the amino terminalconserved end plus the variable region of Flagellin X.

SEQ ID NO:78 is the determined cDNA sequence for the carboxy-terminalconserved end of Flagellin X.

SEQ ID NO:79 is the full-length amino acid sequence of Flagellin X.

SEQ ID NO:80 is the amino acid sequence of the amino terminal conservedend of Flagellin X.

SEQ ID NO:81 is the amino acid sequence of the amino terminal conservedend plus the variable region of Flagellin X.

SEQ ID NO:82 is the amino acid sequence of the carboxy-terminalconserved end of Flagellin X.

SEQ ID NO:83 is the full-length coding sequence of Helicobacter bilisflagellin B.

SEQ ID NO:84 is the full-length protein sequence of Helicobacter bilisflagellin B, encoded by the nucleotide sequence set forth in SEQ IDNO:83.

SEQ ID NO:85 is the full-length coding sequence of Cbir-1 flagellin(partial sequence set forth in SEQ ID NO:1).

SEQ ID NO:86 is the full-length protein sequence of Cbir-1 flagellin,encoded by the nucleotide sequence set forth in SEQ ID NO:85.

SEQ ID NO:87 is the determined full length cDNA sequence for clone 76963(SEQ ID NO:9), CBir-11.

SEQ ID NO:88 is a predicted translated protein sequence encoded by SEQID NO:86, a flagellin-like sequence.

SEQ ID NO:89 is a predicted translated protein sequence encoded by SEQID NO:86, a phosphoesterase-like sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed generally to compositions and theiruse in the therapy and diagnosis of IBD. As described further below,illustrative compositions of the present invention include, but are notrestricted to, polypeptides, particularly immunogenic polypeptides,polynucleotides encoding such polypeptides, antibodies and other bindingagents, antigen presenting cells (APCs) and immune system cells (e.g., Tand B cells).

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. All references cited herein are eachincorporated by reference in their entirety.

Polypeptide Compositions

As used herein, the term “polypeptide” “is used in its conventionalmeaning, i.e., as a sequence of amino acids. The polypeptides are notlimited to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.,antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse.

Particularly illustrative polypeptides of the present invention comprisethose encoded by a polynucleotide sequence set forth in any one of SEQID NOs:1-37, 51-78, 83, 85 and 87, or a sequence that hybridizes undermoderately stringent conditions, or, alternatively, under highlystringent conditions, to a polynucleotide sequence set forth in any oneof SEQ ID NOs:1-37, 51-78, 83, 85 and 87. Certain other illustrativepolypeptides of the invention comprise amino acid sequences as set forthin any one of SEQ ID NOs:38-50, 79-82, 84, and 86.

The polypeptides of the present invention are sometimes herein referredto as bacterial proteins or bacterial polypeptides, as an indicationthat their identification has been based at least in part upon theirexpression in enteric bacterial samples isolated from the colon ofindividuals with IBD. The peptides described herein may be identifiedfrom a lesion in the colon from a patient with IBD. Accordingly, such apeptide may not be present in adjacent normal tissue. Alternatively, apeptide of the present invention may be identified from an entericbacterial sample isolated from the colon of an individual with IBD saidenteric bacteria being absent from individuals not affected with IBD. Ina further embodiment, the polypeptides of the present invention may beidentified by their ability to activate T cells from individualsaffected with IBD. Additionally, polypeptides described herein may beidentified by their reactivity with sera from IBD patients as comparedto their lack of reactivity to sera from unaffected individuals.

Thus, a “bacterial polypeptide” or “bacterial protein,” refers generallyto a polypeptide sequence of the present invention, or a polynucleotidesequence encoding such a polypeptide, that is present in samplesisolated from a substantial proportion of IBD patients, for examplepreferably greater than about 20%, more preferably greater than about30%, and most preferably greater than about 50% or more of patientstested as determined using a representative assay provided herein. Abacterial polypeptide sequence of the invention, based upon itsexpression in enteric bacterial samples isolated from the colon ofindividuals with IBD, has particular utility both as a diagnostic markeras well as a therapeutic target, as further described below. In oneparticular embodiment of the present invention, a bacterial polypeptideor bacterial protein comprises a flagellin protein.

In certain preferred embodiments, the polypeptides of the invention areimmunogenic, i.e., they react detectably within an immunoassay (such asan ELISA or T-cell stimulation assay) with antisera and/or T-cells froma patient with IBD. Screening for immunogenic activity can be performedusing techniques well known to the skilled artisan. For example, suchscreens can be performed using methods such as those described in Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In one illustrative example, a polypeptide may beimmobilized on a solid support and contacted with patient sera to allowbinding of antibodies within the sera to the immobilized polypeptide.Unbound sera may then be removed and bound antibodies detected using,for example, ¹²⁵I-labeled Protein A.

As would be recognized by the skilled artisan, immunogenic portions ofthe polypeptides disclosed herein are also encompassed by the presentinvention. An “immunogenic portion,” as used herein, is a fragment of animmunogenic polypeptide of the invention that itself is immunologicallyreactive (i.e., specifically binds) with the B-cells and/or T-cellsurface antigen receptors that recognize the polypeptide. Immunogenicportions may generally be identified using well known techniques, suchas those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247(Raven Press, 1993) and references cited therein. Such techniquesinclude screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques.

In one preferred embodiment, an immunogenic portion of a polypeptide ofthe present invention is a portion that reacts with antisera and/orT-cells at a level that is not substantially less than the reactivity ofthe full-length polypeptide (e.g., in an ELISA and/or T-cell reactivityassay). Preferably, the level of immunogenic activity of the immunogenicportion is at least about 50%, preferably at least about 70% and mostpreferably greater than about 90% of the immunogenicity for thefull-length polypeptide. In some instances, preferred immunogenicportions will be identified that have a level of immunogenic activitygreater than that of the corresponding full-length polypeptide, e.g.,having greater than about 100% or 150% or more immunogenic activity.

In certain other embodiments, illustrative immunogenic portions mayinclude peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

In another embodiment, a polypeptide composition of the invention mayalso comprise one or more polypeptides that are immunologically reactivewith T cells and/or antibodies generated against a polypeptide of theinvention, particularly a polypeptide having an amino acid sequencedisclosed herein, or to an immunogenic fragment or variant thereof.

In another embodiment of the invention, polypeptides are provided thatcomprise one or more polypeptides that are capable of eliciting T cellsand/or antibodies that are immunologically reactive with one or morepolypeptides described herein, or one or more polypeptides encoded bycontiguous nucleic acid sequences contained in the polynucleotidesequences disclosed herein, or immunogenic fragments or variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency.

The present invention, in another aspect, provides polypeptide fragmentscomprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous aminoacids, or more, including all intermediate lengths, of a polypeptidecompositions set forth herein, such as those set forth in SEQ IDNOs:38-50, 79-82, 84, 86 and 88-89, or those encoded by a polynucleotidesequence set forth in a sequence of SEQ ID NOs:1-37, 51-78, 83, 85 and87.

In another aspect, the present invention provides variants of thepolypeptide compositions described herein. Polypeptide variantsgenerally encompassed by the present invention will typically exhibit atleast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more identity (determined as described below), along itslength, to a polypeptide sequences set forth herein.

In one preferred embodiment, the polypeptide fragments and variantsprovided by the present invention are immunologically reactive with anantibody and/or T-cell that reacts with a full-length polypeptidespecifically set forth herein.

In another preferred embodiment, the polypeptide fragments and variantsprovided by the present invention exhibit a level of immunogenicactivity of at least about 50%, preferably at least about 70%, and mostpreferably at least about 90% or more of that exhibited by a full-lengthpolypeptide sequence specifically set forth herein.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants may be naturally occurring or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating their immunogenicactivity as described herein and/or using any of a number of techniqueswell known in the art.

For example, certain illustrative variants of the polypeptides of theinvention include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other illustrative variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

In many instances, a variant will contain conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. As described above, modifications may be made in thestructure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the invention, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl-methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polypeptide sequences, two sequences are said to be“identical” if the sequence of amino acids in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Saitou, N. Nei, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. For amino acid sequences,a scoring matrix can be used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one preferred approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

Within other illustrative embodiments, a polypeptide may be a fusionpolypeptide that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known bacterial protein. A fusion partnermay, for example, assist in providing T helper epitopes (animmunological fusion partner), preferably T helper epitopes recognizedby humans, or may assist in expressing the protein (an expressionenhancer) at higher yields than the native recombinant protein. Certainpreferred fusion partners are both immunological and expressionenhancing fusion partners. Other fusion partners may be selected so asto increase the solubility of the polypeptide or to enable thepolypeptide to be targeted to desired intracellular compartments. Stillfurther fusion partners include affinity tags, which facilitatepurification of the polypeptide.

Fusion polypeptides may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion polypeptide isexpressed as a recombinant polypeptide, allowing the production ofincreased levels, relative to a non-fused polypeptide, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion polypeptide that retains the biological activity ofboth component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion polypeptideusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

The fusion polypeptide can comprise a polypeptide as described hereintogether with an unrelated immunogenic protein, such as an immunogenicprotein capable of eliciting a recall response. Examples of suchproteins include tetanus, tuberculosis and hepatitis proteins (see, forexample, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derivedfrom a Mycobacterium sp., such as a Mycobacterium tuberculosis-derivedRa12 fragment. Ra12 compositions and methods for their use in enhancingthe expression and/or immunogenicity of heterologouspolynucleotide/polypeptide sequences is described in U.S. PatentApplication 60/158,585, the disclosure of which is incorporated hereinby reference in its entirety. Briefly, Ra12 refers to a polynucleotideregion that is a subsequence of a Mycobacterium tuberculosis MTB32Anucleic acid. MTB32A is a serine protease of 32 KD molecular weightencoded by a gene in virulent and avirulent strains of M. tuberculosis.The nucleotide sequence and amino acid sequence of MTB32A have beendescribed (for example, U.S. Patent Application 60/158,585; see also,Skeiky et al., Infection and Immun. (1999) 67:3998-4007, incorporatedherein by reference). C-terminal fragments of the MTB32A coding sequenceexpress at high levels and remain as a soluble polypeptides throughoutthe purification process. Moreover, Ra12 may enhance the immunogenicityof heterologous immunogenic polypeptides with which it is fused. Onepreferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragmentcorresponding to amino acid residues 192 to 323 of MTB32A. Otherpreferred Ra12 polynucleotides generally comprise at least about 15consecutive nucleotides, at least about 30 nucleotides, at least about60 nucleotides, at least about 100 nucleotides, at least about 200nucleotides, or at least about 300 nucleotides that encode a portion ofa Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence(i.e., an endogenous sequence that encodes a Ra12 polypeptide or aportion thereof) or may comprise a variant of such a sequence. Ra12polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions such that the biological activityof the encoded fusion polypeptide is not substantially diminished,relative to a fusion polypeptide comprising a native Ra12 polypeptide.Variants preferably exhibit at least about 70% identity, more preferablyat least about 80% identity and most preferably at least about 90%identity to a polynucleotide sequence that encodes a native Ra12polypeptide or a portion thereof.

Within other preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

Yet another illustrative embodiment involves fusion polypeptides, andthe polynucleotides encoding them, wherein the fusion partner comprisesa targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class Hmolecules and thereby provide enhanced in vivo stimulation of CD4⁺T-cells specific for the polypeptide.

Polypeptides of the invention are prepared using any of a variety ofwell known synthetic and/or recombinant techniques, the latter of whichare further described below. Polypeptides, portions and other variantsgenerally less than about 150 amino acids can be generated by syntheticmeans, using techniques well known to those of ordinary skill in theart. In one illustrative example, such polypeptides are synthesizedusing any of the commercially available solid-phase techniques, such asthe Merrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

In general, polypeptide compositions (including fusion polypeptides) ofthe invention are isolated. An “isolated” polypeptide is one that isremoved from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

Polynucleotide Compositions

The present invention, in other aspects, provides polynucleotidecompositions. The terms “DNA” and “polynucleotide” are used essentiallyinterchangeably herein to refer to a DNA molecule that has been isolatedfree of total genomic DNA of a particular species. “Isolated,” as usedherein, means that a polynucleotide is substantially away from othercoding sequences, and that the DNA molecule does not contain largeportions of unrelated coding DNA, such as large chromosomal fragments orother functional genes or polypeptide coding regions. Of course, thisrefers to the DNA molecule as originally isolated, and does not excludegenes or coding regions later added to the segment by the hand of man.

As will be understood by those skilled in the art, the polynucleotidecompositions of this invention can include genomic sequences,extra-genomic and plasmid-encoded sequences and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, peptides and the like. Such segments may be naturallyisolated, or modified synthetically by the hand of man.

As will be also recognized by the skilled artisan, polynucleotides ofthe invention may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA or synthetic) or RNAmolecules. RNA molecules may include HnRNA molecules, which containintrons and correspond to a DNA molecule in a one-to-one manner, andmRNA molecules, which do not contain introns. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a polypeptide/protein of the invention or aportion thereof) or may comprise a sequence that encodes a variant orderivative, preferably and immunogenic variant or derivative, of such asequence.

Therefore, according to another aspect of the present invention,polynucleotide compositions are provided that comprise some or all of apolynucleotide sequence set forth in any one of SEQ ID NOs:1-37, 51-78,83, 85 and 87, complements of a polynucleotide sequence set forth in anyone of SEQ ID NOs:1-37, 51-78, 83, 85 and 87, and degenerate variants ofa polynucleotide sequence set forth in any one of SEQ ID NOs:1-37,51-78, 83, 85 and 87. In certain preferred embodiments, thepolynucleotide sequences set forth herein encode immunogenicpolypeptides, as described above. In other certain preferredembodiments, the polynucleotide sequences set forth herein encodeIBD-associated bacterial proteins isolated as described herein fromindividuals affected with IBD. In other certain preferred embodiments,the polynucleotide sequences set forth herein encode flagellin proteins.

In other related embodiments, the present invention providespolynucleotide variants having substantial identity to the sequencesdisclosed herein in SEQ ID NOs:1-37, 51-78, 83, 85, and 87, for examplethose comprising at least 70% sequence identity, preferably at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequenceidentity compared to a polynucleotide sequence of this invention usingthe methods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein). Theterm “variants” should also be understood to encompasses homologousgenes of xenogenic origin.

In additional embodiments, the present invention provides polynucleotidefragments comprising or consisting of various lengths of contiguousstretches of sequence identical to or complementary to one or more ofthe sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise or consist of at least about10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or morecontiguous nucleotides of one or more of the sequences disclosed hereinas well as all intermediate lengths there between. It will be readilyunderstood that “intermediate lengths”, in this context, means anylength between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22,23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103,etc.; 150, 151, 152, 153, etc.; including all integers through 200-500;500-1,000, and the like. A polynucleotide sequence as described here maybe extended at one or both ends by additional nucleotides not found inthe native sequence. This additional sequence may consist of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotidesat either end of the disclosed sequence or at both ends of the disclosedsequence.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS. One skilled in the art will understandthat the stringency of hybridization can be readily manipulated, such asby altering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60-65° C. or 65-70° C.

In certain preferred embodiments, the polynucleotides described above,e.g., polynucleotide variants, fragments and hybridizing sequences,encode polypeptides that are immunologically cross-reactive with apolypeptide sequence specifically set forth herein. In other preferredembodiments, such polynucleotides encode polypeptides that have a levelof immunogenic activity of at least about 50%, preferably at least about70%, and more preferably at least about 90% of that for a polypeptidesequence specifically set forth herein.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e., thewindow size) and multiplying the results by 100 to yield the percentageof sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification, and/or databasesequence comparison).

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, is employed for thepreparation of immunogenic variants and/or derivatives of thepolypeptides described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In another approach for the production of polypeptide variants of thepresent invention, recursive sequence recombination, as described inU.S. Pat. No. 5,837,458, may be employed. In this approach, iterativecycles of recombination and screening or selection are performed to“evolve” individual polynucleotide variants of the invention having, forexample, enhanced immunogenic activity.

In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise or consist of a sequence region ofat least about a 15 nucleotide long contiguous sequence that has thesame sequence as, or is complementary to, a 15 nucleotide longcontiguous sequence disclosed herein will find particular utility.Longer contiguous identical or complementary sequences, e.g., those ofabout 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediatelengths) and even up to full length sequences will also be of use incertain embodiments.

The ability of such nucleic acid probes to specifically hybridize to asequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review thesequences set forth herein, or to any continuous portion of thesequences, from about 15-25 nucleotides in length up to and includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences may be governed byvarious factors. For example, one may wish to employ primers fromtowards the termini of the total sequence.

Small polynucleotide segments or fragments may be readily prepared by,for example, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202, byintroducing selected sequences into recombinant vectors for recombinantproduction, and by other recombinant DNA techniques generally known tothose of skill in the art of molecular biology.

The nucleotide sequences of the invention may be used for their abilityto selectively form duplex molecules with complementary stretches of theentire gene or gene fragments of interest. Depending on the applicationenvisioned, one will typically desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by a salt concentration of fromabout 0.02 M to about 0.15 M salt at temperatures of from about 50° C.to about 70° C. Such selective conditions tolerate little, if any,mismatch between the probe and the template or target strand, and wouldbe particularly suitable for isolating related sequences.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

According to another embodiment of the present invention, polynucleotidecompositions comprising antisense oligonucleotides are provided.Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, provide atherapeutic approach by which a disease can be treated by inhibiting thesynthesis of proteins that contribute to the disease. The efficacy ofantisense oligonucleotides for inhibiting protein synthesis is wellestablished. For example, the synthesis of polygalactauronase and themuscarine type 2 acetylcholine receptor are inhibited by antisenseoligonucleotides directed to their respective mRNA sequences (U.S. Pat.No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al.,Science. 1988 Jun. 10; 240 (4858):1544-6; Vasanthakumar and Ahmed,Cancer Commun. 1989; 1 (4):225-32; Penis et al., Brain Res Mol BrainRes. 1998 Jun. 15; 57 (2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No.5,789,573; U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288).Antisense constructs have also been described that inhibit and can beused to treat a variety of abnormal cellular proliferations, e.g. cancer(U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

Therefore, in certain embodiments, the present invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein. Selection of antisense compositions specific for a given genesequence is based upon analysis of the chosen target sequence anddetermination of secondary structure, T_(m), binding energy, andrelative stability. Antisense compositions may be selected based upontheir relative inability to form dimers, hairpins, or other secondarystructures that would reduce or prohibit specific binding to the targetmRNA in a host cell. Highly preferred target regions of the mRNA, arethose which are at or near the AUG translation initiation codon, andthose sequences which are substantially complementary to 5′ regions ofthe mRNA. These secondary structure analyses and target site selectionconsiderations can be performed, for example, using v.4 of the OLIGOprimer analysis software and/or the BLASTN 2.0.5 algorithm software(Altschul et al., Nucleic Acids Res. 1997, 25 (17):3389-402).

The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 Jul. 15; 25(14):2730-6). It has been demonstrated that several molecules of the MPGpeptide coat the antisense oligonucleotides and can be delivered intocultured mammalian cells in less than 1 hour with relatively highefficiency (90%). Further, the interaction with MPG strongly increasesboth the stability of the oligonucleotide to nuclease and the ability tocross the plasma membrane.

According to another embodiment of the invention, the polynucleotidecompositions described herein are used in the design and preparation ofribozyme molecules for inhibiting expression of the bacterialpolypeptides and proteins of the present invention in bacterial cells.Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA.1987 December; 84 (24):8788-92; Forster and Symons, Cell. 1987 Apr. 24;49 (2):211-20). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Cech et al., Cell. 1981 December; 27 (3 Pt 2):487-96; Micheland Westhof, J Mol Biol. 1990 Dec. 5; 216 (3):585-610; Reinhold-Hurekand Shub, Nature. 1992 May 14; 357 (6374):173-6). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., Proc Natl Acad SciUSA. 1992 Aug. 15; 89 (16):7305-9). Thus, the specificity of action of aribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. NucleicAcids Res. 1992 Sep. 11; 20 (17):4559-65. Examples of hairpin motifs aredescribed by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),Hampel and Tritz, Biochemistry 1989 Jun. 13; 28 (12):4929-33; Hampel etal., Nucleic Acids Res. 1990 Jan. 25; 18 (2):299-304 and U.S. Pat. No.5,631,359. An example of the hepatitis δ virus motif is described byPerrotta and Been, Biochemistry. 1992 Dec. 1; 31 (47):11843-52; anexample of the RNaseP motif is described by Guerrier-Takada et al.,Cell. 1983 December; 35 (3 Pt 2):849-57; Neurospora VS RNA ribozymemotif is described by Collins (Saville and Collins, Cell. 1990 May 18;61 (4):685-96; Saville and Collins, Proc Natl Acad Sci USA. 1991 Oct. 1;88 (19):8826-30; Collins and Olive, Biochemistry. 1993 Mar. 23; 32(11):2795-9); and an example of the Group I intron is described in (U.S.Pat. No. 4,987,071). All that is important in an enzymatic nucleic acidmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595) and synthesized tobe tested in vitro and in vivo, as described. Such ribozymes can also beoptimized for delivery. While specific examples are provided, those inthe art will recognize that equivalent RNA targets in other species canbe utilized when necessary.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells Ribozymes expressed from suchpromoters have been shown to function in mammalian cells. Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

In another embodiment of the invention, peptide nucleic acids (PNAs)compositions are provided. PNA is a DNA mimic in which the nucleobasesare attached to a pseudopeptide backbone (Good and Nielsen, AntisenseNucleic Acid Drug Dev. 1997 7 (4) 431-37). PNA is able to be utilized ina number methods that traditionally have used RNA or DNA. Often PNAsequences perform better in techniques than the corresponding RNA or DNAsequences and have utilities that are not inherent to RNA or DNA. Areview of PNA including methods of making, characteristics of, andmethods of using, is provided by Corey (Trends Biotechnol 1997 June; 15(6):224-9). As such, in certain embodiments, one may prepare PNAsequences that are complementary to one or more portions of the ACE mRNAsequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., Science 1991 Dec. 6; 254(5037):1497-500; Hanvey et al., Science. 1992 Nov. 27; 258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 January; 4(1):5-23). This chemistry has three important consequences: firstly, incontrast to DNA or phosphorothioate oligonucleotides, PNAs are neutralmolecules; secondly, PNAs are achiral, which avoids the need to developa stereoselective synthesis; and thirdly, PNA synthesis uses standardBoc or Fmoc protocols for solid-phase peptide synthesis, although othermethods, including a modified Merrifield method, have been used.

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., Bioorg Med. Chem. 1995 April; 3 (4):437-45).The manual protocol lends itself to the production of chemicallymodified PNAs or the simultaneous synthesis of families of closelyrelated PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography, providing yields and purity ofproduct similar to those observed during the synthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (for example, Norton etal., Bioorg Med Chem. 1995 April; 3 (4):437-45; Petersen et al., J PeptSci. 1995 May-June; 1 (3):175-83; Orum et al., Biotechniques. 1995September; 19 (3):472-80; Footer et al., Biochemistry. 1996 Aug. 20; 35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug. 11; 23(15):3003-8; Pardridge et al., Proc Natl Acad Sci USA. 1995 Jun. 6; 92(12):5592-6; Boffa et al., Proc Natl Acad Sci USA. 1995 Mar. 14; 92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 Aug. 15; 88(4):1411-7; Armitage et al., Proc Natl Acad Sci USA. 1997 Nov. 11; 94(23):12320-5; Seeger et al., Biotechniques. 1997 September; 23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimericmolecules and their uses in diagnostics, modulating protein inorganisms, and treatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (Anal Chem. 1993 Dec. 15; 65 (24):3545-9) and Jensenet al. (Biochemistry. 1997 Apr. 22; 36 (16):5072-7). Rose uses capillarygel electrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology.

Other applications of PNAs that have been described and will be apparentto the skilled artisan include use in DNA strand invasion, antisenseinhibition, mutational analysis, enhancers of transcription, nucleicacid purification, isolation of transcriptionally active genes, blockingof transcription factor binding, genome cleavage, biosensors, in situhybridization, and the like.

Polynucleotide Identification, Characterization and Expression

Polynucleotides compositions of the present invention may be identified,prepared and/or manipulated using any of a variety of well establishedtechniques (see generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989, and other like references). For example, a polynucleotidemay be identified, as described in more detail below, by screening amicroarray of cDNAs for bacterial cDNAs present in tissue samplesisolated from individuals affected with IBD as compared to samplesisolated from unaffected individuals. Such screens may be performed, forexample, using the microarray technology of Affymetrix, Inc. (SantaClara, Calif.) according to the manufacturer's instructions (andessentially as described by Schena et al., Proc. Natl. Acad. Sci. USA93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA94:2150-2155, 1997). Alternatively, polynucleotide compositions of thepresent invention may be identified by screening mouse or human cecalbacteria genomic random shear expression libraries, as described inExample 1.

Alternatively, polynucleotides may be amplified from cDNA prepared fromcells expressing the bacterial proteins described herein. Many templatedependent processes are available to amplify a target sequences ofinterest present in a sample. One of the best known amplificationmethods is the polymerase chain reaction (PCR™) which is described indetail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each ofwhich is incorporated herein by reference in its entirety. Briefly, inPCR™, two primer sequences are prepared which are complementary toregions on opposite complementary strands of the target sequence. Anexcess of deoxynucleoside triphosphates is added to a reaction mixturealong with a DNA polymerase (e.g., Taq polymerase). If the targetsequence is present in a sample, the primers will bind to the target andthe polymerase will cause the primers to be extended along the targetsequence by adding on nucleotides. By raising and lowering thetemperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Any of a number of other template dependent processes, many of which arevariations of the PCR™ amplification technique, are readily known andavailable in the art. Illustratively, some such methods include theligase chain reaction (referred to as LCR), described, for example, inEur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No. 4,883,750; QbetaReplicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880;Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR).Still other amplification methods are described in Great Britain Pat.Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.PCT/US89/01025. Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (PCT Intl. Pat. Appl.Publ. No. WO 88/10315), including nucleic acid sequence basedamplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822describes a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes anucleic acid sequence amplification scheme based on the hybridization ofa promoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otheramplification methods such as “RACE” (Frohman, 1990), and “one-sidedPCR” (Ohara, 1989) are also well-known to those of skill in the art.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., abacterial cDNA library) using well known techniques. Within suchtechniques, a library (cDNA or genomic) is screened using one or morepolynucleotide probes or primers suitable for amplification. Preferably,a library is size-selected to include larger molecules. Random primedlibraries may also be preferred for identifying 5′ and upstream regionsof genes. Genomic libraries are preferred for obtaining introns andextending 5′ sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

Alternatively, amplification techniques, such as those described above,can be useful for obtaining a full length coding sequence from a partialcDNA sequence. One such amplification technique is inverse PCR (seeTriglia et al., Nucl. Acids Res. 16:8186, 1988), which uses restrictionenzymes to generate a fragment in the known region of the gene. Thefragment is then circularized by intramolecular ligation and used as atemplate for PCR with divergent primers derived from the known region.Within an alternative approach, sequences adjacent to a partial sequencemay be retrieved by amplification with a primer to a linker sequence anda primer specific to a known region. The amplified sequences aretypically subjected to a second round of amplification with the samelinker primer and a second primer specific to the known region. Avariation on this procedure, which employs two primers that initiateextension in opposite directions from the known sequence, is describedin WO 96/38591. Another such technique is known as “rapid amplificationof cDNA ends” or RACE. This technique involves the use of an internalprimer and an external primer, which hybridizes to a polyA region orvector sequence, to identify sequences that are 5′ and 3′ of a knownsequence. Additional techniques include capture PCR (Lagerstrom et al.,PCR Methods Applic. 1:111-19, 1991) and walking PCR (Parker et al.,Nucl. Acids. Res. 19:3055-60, 1991). Other methods employingamplification may also be employed to obtain a full length cDNAsequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co., NewYork, N.Y.) or other comparable techniques available in the art. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thepBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, any of a number of expression vectors may beselected depending upon the use intended for the expressed polypeptide.For example, when large quantities are needed, for example for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as pBLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of .beta.-galactosidase so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Natl. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation.glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) geneswhich can be employed in tk.sup.- or aprt.sup.-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). Theuse of visible markers has gained popularity with such markers asanthocyanins, beta-glucuronidase and its substrate GUS, and luciferaseand its substrate luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes, C.A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells that contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include, for example, membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

Antibody Compositions, Fragments Thereof and Other Binding Agents

According to another aspect, the present invention further providesbinding agents, such as antibodies and antigen-binding fragmentsthereof, that exhibit immunological binding to a bacterial polypeptidedisclosed herein, or to a portion, variant or derivative thereof. In oneparticular embodiment, the antibodies of the present invention bind to aToll-like receptor. Illustrative Toll-like receptors (TLR) include, butare not limited to, TLR5. In a related embodiment, the antibodies of thepresent invention may bind to a flagellin protein. In anotherembodiment, the antibodies of the present invention are neutralizingantibodies that block the interaction between TLR5 and a flagellinprotein.

An antibody, or antigen-binding fragment thereof, is said to“specifically bind,” “immunogically bind,” and/or is “immunologicallyreactive” to a polypeptide of the invention if it reacts at a detectablelevel (within, for example, an ELISA assay) with the polypeptide, anddoes not react detectably with unrelated polypeptides under similarconditions.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem.59:439-473.

An “antigen-binding site,” or “binding portion” of an antibody refers tothe part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs”. Thus the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

Binding agents may be further capable of differentiating betweenpatients with and without IBD, using the representative assays providedherein. For example, antibodies or other binding agents that bind to abacterial protein will preferably generate a signal indicating thepresence of IBD in at least about 20% of patients with the disease, morepreferably at least about 30% of patients. Alternatively, or inaddition, the antibody will generate a negative signal indicating theabsence of the disease in at least about 90% of individuals without IBD.To determine whether a binding agent satisfies this requirement,biological samples (e.g., blood, sera, sputum, urine, feces, and/orbiopsies) from patients with and without IBD (as determined usingstandard clinical tests) may be assayed as described herein for thepresence of polypeptides that bind to the binding agent. Preferably, astatistically significant number of samples with and without the diseasewill be assayed. Each binding agent should satisfy the above criteria;however, those of ordinary skill in the art will recognize that bindingagents may be used in combination to improve sensitivity.

Binding agents may be further capable of identifying patients at riskfor developing IBD, using the representative assays provided herein. Forexample, antibodies or other binding agents that bind to a bacterialprotein will preferably generate a signal indicating a risk for thedevelopment of IBD in at least about 20% of patients with positivefamily history of the disease, or patients with a defined genetic riskfor developing the disease (for example, individuals positive for theNOD2 mutation), more preferably at least about 30% of said individuals.Alternatively, or in addition, the antibody will generate a negativesignal indicating the absence of risk for developing disease in at leastabout 90% of individuals with no family history or with no definedgenetic risk of developing IBD. To determine whether a binding agentsatisfies this requirement, biological samples (e.g., blood, sera,sputum, urine, feces, and/or biopsies) from patients with and withoutrisk factors for the development of IBD (as determined using standardclinical tests) may be assayed as described herein for the presence ofpolypeptides that bind to the binding agent. Preferably, a statisticallysignificant number of samples from individuals with and without risk ofdeveloping the disease will be assayed. Each binding agent shouldsatisfy the above criteria; however, those of ordinary skill in the artwill recognize that binding agents may be used in combination to improvesensitivity.

Binding agents of the present invention may be further used, eitheralone or in combination with other diagnostic modalities, to subdivideIBD patients into categories of disease that would be susceptible orresistant to new or existing treatments. In particular, serum reactivityagainst the binding agents to subdivide IBD patients would be useful asa stand alone diagnostic or in combination with other known diagnosticmarkers and assays, such as pANCA. Such binding agents could also beused to distinguish clinical subgroups of patients with Crohn's Disease,which would be of relevance to predict the clinical course of anindividual patient or predict responsivensess to particular medication.Diagnostic use may be as a stand-alone test or used in combination withother serological testing methods or in combination with standardlaboratory, clinical or pathological testing or with DNA-based testingsuch as for NOD2 mutations.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85 (16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRS and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRS. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

As used herein, the terms “veneered FRs” and “recombinantly veneeredFRs” refer to the selective replacement of FR residues from, e.g., arodent heavy or light chain V region, with human FR residues in order toprovide a xenogeneic molecule comprising an antigen-binding site whichretains substantially all of the native FR polypeptide foldingstructure. Veneering techniques are based on the understanding that theligand binding characteristics of an antigen-binding site are determinedprimarily by the structure and relative disposition of the heavy andlight chain CDR sets within the antigen-binding surface. Davies et al.(1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificitycan be preserved in a humanized antibody only wherein the CDRstructures, their interaction with each other, and their interactionwith the rest of the V region domains are carefully maintained. By usingveneering techniques, exterior (e.g., solvent-accessible) FR residueswhich are readily encountered by the immune system are selectivelyreplaced with human residues to provide a hybrid molecule that compriseseither a weakly immunogenic, or substantially non-immunogenic veneeredsurface.

The process of veneering makes use of the available sequence data forhuman antibody variable domains compiled by Kabat et al., in Sequencesof Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Healthand Human Services, U.S. Government Printing Office, 1987), updates tothe Kabat database, and other accessible U.S. and foreign databases(both nucleic acid and protein). Solvent accessibilities of V regionamino acids can be deduced from the known three-dimensional structurefor human and murine antibody fragments. There are two general steps inveneering a murine antigen-binding site. Initially, the FRs of thevariable domains of an antibody molecule of interest are compared withcorresponding FR sequences of human variable domains obtained from theabove-identified sources. The most homologous human V regions are thencompared residue by residue to corresponding murine amino acids. Theresidues in the murine FR which differ from the human counterpart arereplaced by the residues present in the human moiety using recombinanttechniques well known in the art. Residue switching is only carried outwith moieties which are at least partially exposed (solvent accessible),and care is exercised in the replacement of amino acid residues whichmay have a significant effect on the tertiary structure of V regiondomains, such as proline, glycine and charged amino acids.

In this manner, the resultant “veneered” murine antigen-binding sitesare thus designed to retain the murine CDR residues, the residuessubstantially adjacent to the CDRs, the residues identified as buried ormostly buried (solvent inaccessible), the residues believed toparticipate in non-covalent (e.g., electrostatic and hydrophobic)contacts between heavy and light chain domains, and the residues fromconserved structural regions of the FRs which are believed to influencethe “canonical” tertiary structures of the CDR loops. These designcriteria are then used to prepare recombinant nucleotide sequences whichcombine the CDRs of both the heavy and light chain of a murineantigen-binding site into human-appearing FRs that can be used totransfect mammalian cells for the expression of recombinant humanantibodies which exhibit the antigen specificity of the murine antibodymolecule.

In another embodiment of the invention, monoclonal antibodies of thepresent invention may be coupled to one or more therapeutic agents.Suitable agents in this regard include radionuclides, differentiationinducers, drugs, toxins, and derivatives thereof. Preferredradionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato etal.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat.No. 4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562, to Davison et al. discloses representativechelating compounds and their synthesis.

T Cell Compositions

The present invention, in another aspect, provides T cells specific fora bacterial polypeptide disclosed herein, or for a variant or derivativethereof. Such cells may generally be prepared in vitro or ex vivo, usingstandard procedures. For example, T cells may be isolated from biopsies,bone marrow, peripheral blood, or a fraction of bone marrow orperipheral blood of a patient, using a commercially available cellseparation system, such as the Isolex™ System, available from NexellTherapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856;U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Inone particular embodiment of the present invention, T cells may beisolated from intraepithelial lymphocytes (IEL) or lamina proprialymphocyte (LPL) samples originating from colon biopsies. Individualswith skill in the art will readily recognize that there numerousmethodologies for isolating IEL and LPL (for example, methods describedin Christ, A. D., S. P. Colgan, S. P. Balk, R. S. Blumberg. 1997.Immunol. Lett. 58:159; Boll G, Reimann J. Scand J Immunol 1995 August;42 (2):191-201). In certain aspects, T cells may be derived from relatedor unrelated humans, non-human mammals, cell lines or cultures.

T cells may be stimulated with a polypeptide, polynucleotide encoding apolypeptide and/or an antigen presenting cell (APC) that expresses sucha polypeptide. Such stimulation is performed under conditions and for atime sufficient to permit the generation of T cells that are specificfor the polypeptide of interest. Preferably, a bacterial polypeptide orpolynucleotide of the invention is present within a delivery vehicle,such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the presentinvention if the T cells specifically proliferate, secrete cytokines orkill target cells coated with the polypeptide or expressing a geneencoding the polypeptide. T cell specificity may be evaluated using anyof a variety of standard techniques. For example, within a chromiumrelease assay or proliferation assay, a stimulation index of more thantwo fold increase in lysis and/or proliferation, compared to negativecontrols, indicates T cell specificity. Such assays may be performed,for example, as described in Chen et al., Cancer Res. 54:1065-1070,1994. Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a bacterial polypeptide (100 ng/ml-100 μg/ml,preferably 200 ng/ml-25 μg/ml) for 3-7 days will typically result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells thathave been activated in response to a bacterial polypeptide,polynucleotide or polypeptide-expressing APC may be CD4⁺ and/or CD8⁺.Bacterial polypeptide-specific T cells may be expanded using standardtechniques. Within preferred embodiments, the T cells are derived from apatient, a related donor or an unrelated donor, and are administered tothe patient following stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a bacterial polypeptide, polynucleotide or APC can beexpanded, in number either in vitro or in vivo. Proliferation of such Tcells in vitro may be accomplished in a variety of ways. For example,the T cells can be re-exposed to a bacterial polypeptide, or a shortpeptide corresponding to an immunogenic portion of such a polypeptide,with or without the addition of T cell growth factors, such asinterleukin-2, and/or stimulator cells that synthesize a bacterialpolypeptide. Alternatively, one or more T cells that proliferate in thepresence of the bacterial polypeptide can be expanded in number bycloning. Methods for cloning cells are well known in the art, andinclude limiting dilution.

In certain embodiments, T cells that produce anti-inflammatory cytokinesmay be desirable. Such cytokines may include, but are not limited to, 10(IL-10), interferon-γ (IFN-γ), interleukin 4 (IL-4), interleukin 12(IL-12), transforming growth factor beta (TGF

and interleukin 18 (IL-18). In certain embodiments, an anti-inflammatoryresponse is mediated by CD4+ T helper cells.

T Cell Receptor Compositions

The T cell receptor (TCR) consists of 2 different, highly variablepolypeptide chains, termed the T-cell receptor α and β chains, that arelinked by a disulfide bond (Janeway, Travers, Walport. Immunobiology.Fourth Ed., 148-159. Elsevier Science Ltd/Garland Publishing. 1999). Theα/β heterodimer complexes with the invariant CD3 chains at the cellmembrane. This complex recognizes specific antigenic peptides bound toMHC molecules. The enormous diversity of TCR specificities is generatedmuch like immunoglobulin diversity, through somatic gene rearrangement.The β chain genes contain over 50 variable (V), 2 diversity (D), over 10joining (J) segments, and 2 constant region segments (C). The α chaingenes contain over 70 V segments, and over 60 J segments but no Dsegments, as well as one C segment. During T cell development in thethymus, the D to J gene rearrangement of the β chain occurs, followed bythe V gene segment rearrangement to the DJ. This functional VDJ_(β) exonis transcribed and spliced to join to a C_(β). For the α chain, a V_(α)gene segment rearranges to a J_(α) gene segment to create the functionalexon that is then transcribed and spliced to the C_(α). Diversity isfurther increased during the recombination process by the randomaddition of P and N-nucleotides between the V, D, and J segments of theβ chain and between the V and J segments in the α chain (Janeway,Travers, Walport. Immunobiology. Fourth Ed., 98 and 150. ElsevierScience Ltd/Garland Publishing. 1999).

The present invention, in another aspect, provides TCRs specific for apolypeptide disclosed herein, or for a variant or derivative thereof. Inaccordance with the present invention, polynucleotide and amino acidsequences are provided for the V-J or V-D-J junctional regions or partsthereof for the alpha and beta chains of the T-cell receptor whichrecognize bacterial polypeptides described herein. In general, thisaspect of the invention relates to T-cell receptors which recognize orbind bacterial polypeptides presented in the context of MHC. In apreferred embodiment the bacterial antigens recognized by the T-cellreceptors comprise a polypeptide of the present invention. For example,cDNA encoding a TCR specific for a bacterial peptide can be isolatedfrom T cells specific for a bacterial polypeptide using standardmolecular biological and recombinant DNA techniques.

This invention further includes the T-cell receptors or analogs thereofhaving substantially the same function or activity as the T-cellreceptors of this invention which recognize or bind bacterialpolypeptides. Such receptors include, but are not limited to, a fragmentof the receptor, or a substitution, addition or deletion mutant of aT-cell receptor provided herein. This invention also encompassespolypeptides or peptides that are substantially homologous to the T-cellreceptors provided herein or that retain substantially the sameactivity. The term “analog” includes any protein or polypeptide havingan amino acid residue sequence substantially identical to the T-cellreceptors provided herein in which one or more residues, preferably nomore than 5 residues, more preferably no more than 25 residues have beenconservatively substituted with a functionally similar residue and whichdisplays the functional aspects of the T-cell receptor as describedherein.

The present invention further provides for suitable mammalian hostcells, for example, non-specific T cells, that are transfected with apolynucleotide encoding TCRs specific for a polypeptide describedherein, thereby rendering the host cell specific for the polypeptide.The α and β chains of the TCR may be contained on separate expressionvectors or alternatively, on a single expression vector that alsocontains an internal ribosome entry site (IRES) for cap-independenttranslation of the gene downstream of the IRES. Said host cellsexpressing TCRs specific for the polypeptide may be used, for example,for adoptive immunotherapy of IBD as discussed further below.

In further aspects of the present invention, cloned TCRs specific for apolypeptide recited herein may be used in a kit for the diagnosis ofIBD. For example, the nucleic acid sequence or portions thereof, ofbacterial-specific TCRs can be used as probes or primers for thedetection of expression of the rearranged genes encoding the specificTCR in a biological sample. Therefore, the present invention furtherprovides for an assay for detecting messenger RNA or DNA encoding theTCR specific for a polypeptide.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell, TCR, and/orantibody compositions disclosed herein in pharmaceutically-acceptablecarriers for administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy.

It will be understood that, if desired, a composition as disclosedherein may, be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Therefore, in another aspect of the present invention, pharmaceuticalcompositions are provided comprising one or more of the polynucleotide,polypeptide, antibody, TCR, and/or T-cell compositions described hereinin combination with a physiologically acceptable carrier. In certainpreferred embodiments, the pharmaceutical compositions of the inventioncomprise immunogenic polynucleotide and/or polypeptide compositions ofthe invention for use in prophylactic and therapeutic vaccineapplications. Vaccine preparation is generally described in, forexample, M. F. Powell and M. J. Newman, eds., “Vaccine Design (thesubunit and adjuvant approach),” Plenum Press (NY, 1995). Generally,such compositions will comprise one or more polynucleotide and/orpolypeptide compositions of the present invention in combination withone or more immunostimulants.

It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable salts of thepolynucleotides and polypeptides of the invention. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g., salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g., sodium,potassium, lithium, ammonium, calcium and magnesium salts).

In another embodiment, illustrative immunogenic compositions, e.g.,vaccine compositions, of the present invention comprise DNA encoding oneor more of the polypeptides as described above, such that thepolypeptide is generated in situ. As noted above, the polynucleotide maybe administered within any of a variety of delivery systems known tothose of ordinary skill in the art. Indeed, numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, andreferences cited therein. Appropriate polynucleotide expression systemswill, of course, contain the necessary regulatory DNA regulatorysequences for expression in a patient (such as a suitable promoter andterminating signal). Alternatively, bacterial delivery systems mayinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

Therefore, in certain embodiments, polynucleotides encoding immunogenicpolypeptides described herein are introduced into suitable mammalianhost cells for expression using any of a number of known viral-basedsystems. In one illustrative embodiment, retroviruses provide aconvenient and effective platform for gene delivery systems. A selectednucleotide sequence encoding a polypeptide of the present invention canbe inserted into a vector and packaged in retroviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to a subject. A number of illustrative retroviral systemshave been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman(1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin(1993) Cur. Opin. Genet. Develop. 3:102-109.

In addition, a number of illustrative adenovirus-based systems have alsobeen described. Unlike retroviruses which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Additional viral vectors useful for delivering the polynucleotidesencoding polypeptides of the present invention by gene transfer includethose derived from the pox family of viruses, such as vaccinia virus andavian poxvirus. By way of example, vaccinia virus recombinantsexpressing the novel molecules can be constructed as follows. The DNAencoding a polypeptide is first inserted into an appropriate vector sothat it is adjacent to a vaccinia promoter and flanking vaccinia DNAsequences, such as the sequence encoding thymidine kinase (TK). Thisvector is then used to transfect cells which are simultaneously infectedwith vaccinia. Homologous recombination serves to insert the vacciniapromoter plus the gene encoding the polypeptide of interest into theviral genome. The resulting TK.sup.(−) recombinant can be selected byculturing the cells in the presence of 5-bromodeoxyuridine and pickingviral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently usedto provide for inducible, transient expression or coexpression of one ormore polypeptides described herein in host cells of an organism. In thisparticular system, cells are first infected in vitro with a vacciniavirus recombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide or polynucleotides of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into polypeptide by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used for delivery ofpolynucleotide compositions of the present invention, such as thosevectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE)can also be used, illustrative examples of which can be found in U.S.Pat. Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as the adenovirus chimericvectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, canalso be used for gene delivery under the invention.

Additional illustrative information on these and other known viral-baseddelivery systems can be found, for example, in Fisher-Hoch et al., Proc.Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad.Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993.

In certain embodiments, a polynucleotide may be integrated into thegenome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

In still another embodiment, a composition of the present invention canbe delivered via a particle bombardment approach, many of which havebeen described. In one illustrative example, gas-driven particleacceleration can be achieved with devices such as those manufactured byPowderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc.(Madison, Wis.), some examples of which are described in U.S. Pat. Nos.5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799.This approach offers a needle-free delivery approach wherein a drypowder formulation of microscopic particles, such as polynucleotide orpolypeptide particles, are accelerated to high speed within a helium gasjet generated by a hand held device, propelling the particles into atarget tissue of interest.

In a related embodiment, other devices and methods that may be usefulfor gas-driven needle-less injection of compositions of the presentinvention include those provided by Bioject, Inc. (Portland, Oreg.),some examples of which are described in U.S. Pat. Nos. 4,790,824;5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

According to another embodiment, the pharmaceutical compositionsdescribed herein will comprise one or more immunostimulants in additionto the immunogenic polynucleotide, polypeptide, antibody, T-cell, TCR,and/or APC compositions of this invention. An immunostimulant refers toessentially any substance that enhances or potentiates an immuneresponse (antibody and/or cell-mediated) to an exogenous antigen. Onepreferred type of immunostimulant comprises an adjuvant. Many adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Certain adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an anti-inflammatory immune response(antibody or cell-mediated). Accordingly, high levels ofanti-inflammatory cytokines (anti-inflammatory cytokines may include,but are not limited to, interleukin 4 (IL-4), interleukin 5 (IL-5),interleukin 10 (IL-10), and transforming growth factor beta (TGF

would be preferred. In certain embodiments, an anti-inflammatoryresponse would be mediated by CD4+ T helper cells. Bacterial flagellinhave been suggested to act as adjunvants (McSorley et al., J. Immunol.169:3914-19, 2002). Within one embodiment of the invention, theflagellin proteins disclosed herein can be used in adjuvantcompositions.

Within other embodiments, the adjuvants used in conjunction with thecompositions of the present invention increase lipopolysaccharide (LPS)responsiveness. Illustrative adjuvants include but are not limited to,monophosphoryl lipid A (MPL), aminoalkyl glucosaminide 4-phosphates(AGPs), including RC-512, RC-522, RC-527, RC-529, RC-544, and RC-560(Corixa, Hamilton, Mont.) and other AGPs such as those described inpending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, thedisclosures of which are incorporated herein by reference in theirentireties.

Within other embodiments of the invention, the adjuvant composition isone that induces an immune response predominantly of the Th1 type. Highlevels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL-12) tend tofavor the induction of cell mediated immune responses to an administeredantigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4,IL-5, IL-6 and IL-10) tend to favor the induction of humoral immuneresponses. Following application of a vaccine as provided herein, apatient will support an immune response that includes Th1- and Th2-typeresponses. Within a preferred embodiment, in which a response ispredominantly Th1-type, the level of Th1-type cytokines will increase toa greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989. Alternatively, in a related embodiment, inwhich a preferred response is predominantly Th2-type, the level ofTh2-type cytokines will increase to a greater extent than the level ofTh1-type cytokines. Again, the levels of these cytokines may be readilyassessed using standard assays.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from Corixa Corporation(Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol® toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), ME-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformula

HO(CH₂CH₂O)_(n)-A-R,  (I):

wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-bacterial effects perse and/or to be immunologically compatible with the receiver (i.e.,matched HLA haplotype). APCs may generally be isolated from any of avariety of biological fluids and organs, including bacterial andperibacterial tissues, and may be autologous, allogeneic, syngeneic orxenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantibacterial immunity (see Timmerman and Levy, Ann. Rev. Med.50:507-529, 1999). In general, dendritic cells may be identified basedon their typical shape (stellate in situ, with marked cytoplasmicprocesses (dendrites) visible in vitro), their ability to take up,process and present antigens with high efficiency and their ability toactivate naïve T cell responses. Dendritic cells may, of course, beengineered to express specific cell-surface receptors or ligands thatare not commonly found on dendritic cells in vivo or ex vivo, and suchmodified dendritic cells are contemplated by the present invention. Asan alternative to dendritic cells, secreted vesicles antigen-loadeddendritic cells (called exosomes) may be used within a vaccine (seeZitvogel et al., Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide of the invention(or portion or other variant thereof) such that the encoded polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the bacterial polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will typically vary depending on the mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems. such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

In another illustrative embodiment, calcium phosphate core particles areemployed as carriers, vaccine adjuvants, or as controlled releasematrices for the compositions of this invention. Exemplary calciumphosphate particles are disclosed, for example, in published patentapplication No. WO/0046147.

The pharmaceutical compositions of the invention will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (see, for example,Mathiowitz et al., Nature 1997 Mar. 27; 386 (6623):410-4; Hwang et al.,Crit Rev Ther Drug Carrier Syst 1998; 15 (3):243-84; U.S. Pat. No.5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations will contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. Alternatively, the active ingredientmay be incorporated into an oral solution such as one containing sodiumborate, glycerin and potassium bicarbonate, or dispersed in adentifrice, or added in a therapeutically-effective amount to acomposition that may include water, binders, abrasives, flavoringagents, foaming agents, and humectants. Alternatively the compositionsmay be fashioned into a tablet or solution form that may be placed underthe tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationswill of course preferably meet sterility, pyrogenicity, and the generalsafety and purity standards as required by FDA Office of Biologicsstandards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., J Controlled Release 1998 Mar. 2; 52 (1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July; 16 (7):307-21;Takakura, Nippon Rinsho 1998 March; 56 (3):691-5; Chandran et al.,Indian J Exp Biol. 1997 August; 35 (8):801-9; Margalit, Crit Rev TherDrug Carrier Syst. 1995; 12 (2-3):233-61; U.S. Pat. No. 5,567,434; U.S.Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 andU.S. Pat. No. 5,795,587, each specifically incorporated herein byreference in its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol Chem. 1990 Sep. 25; 265 (27):16337-42; Muller et al., DNACell Biol. 1990 April; 9 (3):221-9). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes,various drugs, radiotherapeutic agents, enzymes, viruses, transcriptionfactors, allosteric effectors and the like, into a variety of culturedcell lines and animals. Furthermore, he use of liposomes does not appearto be associated with autoimmune responses or unacceptable toxicityafter systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December; 24 (12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit. Rev Ther Drug Carrier Syst. 1988; 5(0:1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March; 45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan. 2; 50(1-3):31-40; and U.S. Pat. No. 5,145,684.

Therapeutic Methods for IBD

Immunologic approaches to IBD therapy are based on the recognition thatIBD represents an “abnormal” mucosal immune response to bacteria withinthe lumen of the gastrointestinal tract. The precise molecular nature ofthe bacterial antigen(s) recognized by the immune system has not beendescribed.

IBD immunotherapy generally focuses on inducing humoral immuneresponses, cellular immune responses, or both, with the goal of inducingtolerance to a particular enteric bacterial antigen, thereby leading toa decrease in inflammation in the gut. Moreover, induction of CD4⁺ Thelper cells is necessary in order to secondarily induce eitherantibodies or cytotoxic CD8⁺ T cells. Polypeptide antigens that areselective or ideally specific to IBD-associated bacteria offer apowerful approach for inducing anti-inflammatory immune responses thateither prevent or ameliorate an aberrant immune response to bacterialantigens associated with IBD, and are an important aspect of the presentinvention.

Therefore, in further aspects of the present invention, thepharmaceutical compositions described herein may be used to stimulate animmune response against bacterial antigens associated with IBD. In oneembodiment of the present invention, the immune response inducedcomprises antibodies that block the interaction of a bacterial antigenwith a host receptor. In one particular embodiment, antibodies inducedby the compositions of the present invention block the interactionbetween flagellin and TLR5, thereby ameliorating the pro-inflammatorycascade initiated by NFKB activation. Alternatively, the compositions ofthe present invention would induce antibodies that stimulateresponsiveness to LPS that ameliorated the hypo-responsiveness inindividuals with Nod2 gene mutation associated with IBD.

In a further embodiment of the present invention, an immune responsewould be anti-inflammatory in nature. For example, a cellular immuneresponse wherein the T cells produce anti-inflammatory cytokines.Anti-inflammatory cytokines may include, but are not limited to, IL-4,IL-5, IL-10, TGF-β.

Within such methods, the pharmaceutical compositions described hereinare administered to a patient, typically a warm-blooded animal,preferably a human. A patient may or may not be afflicted with IBD. Asdiscussed above, administration of the pharmaceutical compositions maybe by any suitable method, including administration by intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal,anal, vaginal, topical and oral routes.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against bacteria with the administration ofimmune response-modifying agents or immunomodulators (such aspolypeptides and polynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedantibacterial immune reactivity (such as effector cells or antibodies)that can directly or indirectly mediate antibacterial effects and doesnot necessarily depend on an intact host immune system. Examples ofeffector cells include T cells as discussed above, T lymphocytes (suchas CD8⁺ cytotoxic T lymphocytes and CD4⁺ T-helper lymphocytes), killercells (such as Natural Killer cells and lymphokine-activated killercells), B cells and antigen-presenting cells (such as dendritic cellsand macrophages) expressing a polypeptide provided herein. T cellreceptors and antibody receptors specific for the polypeptides recitedherein may be cloned, expressed and transferred into other vectors oreffector cells for adoptive immunotherapy. The polypeptides providedherein may also be used to generate antibodies or anti-idiotypicantibodies (as described above and in U.S. Pat. No. 4,918,164) forpassive immunotherapy.

Monoclonal antibodies may be labeled with any of a variety of labels fordesired selective usages in detection, diagnostic assays or therapeuticapplications (as described in U.S. Pat. Nos. 6,090,365; 6,015,542;5,843,398; 5,595,721; and 4,708,930, hereby incorporated by reference intheir entirety as if each was incorporated individually). In each case,the binding of the labelled monoclonal antibody to the determinant siteof the antigen will signal detection or delivery of a particulartherapeutic agent to the antigenic determinant on the non-normal cell. Afurther object of this invention is to provide the specific monoclonalantibody suitably labelled for achieving such desired selective usagesthereof.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal administration.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an appropriate anti-bacterialimmune response, and is at least 10-50% above the basal (i.e.,untreated) level. Alternatively, a suitable dose is an amount of acompound that, when administered as described above, is capable ofdecreasing inflammation in the colon associated with IBD. Such adecrease is at least 10-50% below the untreated level. Such response canbe monitored by measuring the anti-bacterial antibodies in a patient orby vaccine-dependent generation of cytolytic effector cells capable ofrecognizing in vitro bacterial antigens identified from bacteriaisolated from the patient. Such vaccines should also be capable ofcausing an immune response that leads to an improved clinical outcome(e.g., more frequent remissions, complete or partial or longerdisease-free survival) in vaccinated patients as compared tonon-vaccinated patients. In general, for pharmaceutical compositions andvaccines comprising one or more polypeptides, the amount of eachpolypeptide present in a dose ranges from about 25 μg to 5 mg per kg ofhost. Suitable dose sizes will vary with the size of the patient, butwill typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., a decrease in inflammation in thegut, decrease in diarrhea, decrease in steroid requirements orrequirement for other immunosuppressive therapies, decrease in anemia,or decrease in Crohn's disease activity index (CDAI)) in treatedpatients as compared to non-treated patients. Increases in appropriateanti-inflammatory immune responses to a bacterial protein generallycorrelate with an improved clinical outcome. Such immune responses maygenerally be evaluated using standard proliferation, cytotoxicity orcytokine assays, which may be performed using samples obtained from apatient before and after treatment.

Detection and Diagnostic Compositions, Methods and Kits

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In general, the presence or absenceof an IBD-associated bacteria in a patient may be determined by (a)contacting a biological sample obtained from a patient with a bindingagent; (b) detecting in the sample a level of polypeptide that binds tothe binding agent; and (c) comparing the level of polypeptide with apredetermined cut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the polypeptidefrom the remainder of the sample. The bound polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length bacterial proteins and polypeptide portions thereof to whichthe binding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the bacterial protein may be attached. For example,the solid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that polypeptides within the sample are allowed tobind to the immobilized antibody. Unbound sample is then removed fromthe immobilized polypeptide-antibody complexes and a detection reagent(preferably a second antibody capable of binding to a different site onthe polypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of an IBD-associated bacterialpolypeptide within a sample obtained from an individual with IBD atleast about 95% of that achieved at equilibrium between bound andunbound polypeptide. Those of ordinary skill in the art will recognizethat the time necessary to achieve equilibrium may be readily determinedby assaying the level of binding that occurs over a period of time. Atroom temperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of IBD, the signal detected fromthe reporter group that remains bound to the solid support is generallycompared to a signal that corresponds to a predetermined cut-off value.In one preferred embodiment, the cut-off value for the detection of anIBD-associated bacterial antigen is the average mean signal obtainedwhen the immobilized antibody is incubated with samples from patientswithout IBD. In general, a sample generating a signal that is threestandard deviations above the predetermined cut-off value is consideredpositive for IBD-associated bacterial antigens. In an alternatepreferred embodiment, the cut-off value is determined using a ReceiverOperator Curve, according to the method of Sackett et al., ClinicalEpidemiology: A Basic Science for Clinical Medicine, Little Brown andCo., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value maybe determined from a plot of pairs of true positive rates (i.e.,sensitivity) and false positive rates (100%-specificity) that correspondto each possible cut-off value for the diagnostic test result. Thecut-off value on the plot that is the closest to the upper left-handcorner (i.e., the value that encloses the largest area) is the mostaccurate cut-off value, and a sample generating a signal that is higherthan the cut-off value determined by this method may be consideredpositive. Alternatively, the cut-off value may be shifted to the leftalong the plot, to minimize the false positive rate, or to the right, tominimize the false negative rate. In general, a sample generating asignal that is higher than the cut-off value determined by this methodis considered positive for an IBD-associated bacteria.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of an IBD-associated bacterial antigen. Typically, theconcentration of second binding agent at that site generates a pattern,such as a line, that can be read visually. The absence of such a patternindicates a negative result. In general, the amount of binding agentimmobilized on the membrane is selected to generate a visuallydiscernible pattern when the biological sample contains a level ofpolypeptide that would be sufficient to generate a positive signal inthe two-antibody sandwich assay, in the format discussed above.Preferred binding agents for use in such assays are antibodies andantigen-binding fragments thereof. Preferably, the amount of antibodyimmobilized on the membrane ranges from about 25 ng to about 1 μg, andmore preferably from about 50 ng to about 500 ng. Such tests cantypically be performed with a very small amount of biological sample.

Of course, numerous other assay protocols exist that are suitable foruse with the bacterial proteins or binding agents of the presentinvention. The above descriptions are intended to be exemplary only. Forexample, it will be apparent to those of ordinary skill in the art thatthe above protocols may be readily modified to use bacterialpolypeptides to detect antibodies that bind to such polypeptides in abiological sample. The detection of such bacterial protein specificantibodies may correlate with the presence of an IBD-associated antigen.

IBD-associated bacteria may also, or alternatively, be detected based onthe presence of T cells that specifically react with a bacterial proteinin a biological sample. Within certain methods, a biological samplecomprising CD4⁺ and/or CD8⁺ T cells isolated from a patient is incubatedwith a bacterial polypeptide, a polynucleotide encoding such apolypeptide and/or an APC that expresses at least an immunogenic portionof such a polypeptide, and the presence or absence of specificactivation of the T cells is detected. Suitable biological samplesinclude, but are not limited to, isolated T cells. For example, T cellsmay be isolated from a patient by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes). In one particular embodiment of the present invention, Tcells may be isolated from intraepithelial lymphocytes (IEL) or laminapropria lymphocyte (LPL) samples originating from colon biopsies. Tcells may be incubated in vitro for 2-9 days (typically 4 days) at 37□Cwith polypeptide (e.g., 5-25 μg/ml). It may be desirable to incubateanother aliquot of a T cell sample in the absence of bacterialpolypeptide to serve as a control. For CD4⁺ T cells, activation ispreferably detected by evaluating proliferation of the T cells. For CD8⁺T cells, activation is preferably detected by evaluating cytolyticactivity. A level of proliferation that is at least two fold greaterand/or a level of cytolytic activity that is at least 20% greater thanin disease-free patients indicates the presence of IBD-associatedbacteria in the patient.

As noted above, IBD may also, or alternatively, be detected based on thelevel of mRNA encoding a bacterial protein in a biological sample. Forexample, at least two oligonucleotide primers may be employed in apolymerase chain reaction (PCR) based assay to amplify a portion of abacterial cDNA derived from a biological sample, wherein at least one ofthe oligonucleotide primers is specific for (i.e., hybridizes to) apolynucleotide encoding the bacterial protein. The amplified cDNA isthen separated and detected using techniques well known in the art, suchas gel electrophoresis.

Similarly, oligonucleotide probes that specifically hybridize to apolynucleotide encoding a bacterial protein may be used in ahybridization assay to detect the presence of polynucleotide encodingthe bacterial protein in a biological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding abacterial protein of the invention that is at least 10 nucleotides, andpreferably at least 20 nucleotides, in length. Preferably,oligonucleotide primers and/or probes hybridize to a polynucleotideencoding a polypeptide described herein under moderately stringentconditions, as defined above. Oligonucleotide primers and/or probeswhich may be usefully employed in the diagnostic methods describedherein preferably are at least 10-40 nucleotides in length. In apreferred embodiment, the oligonucleotide primers comprise at least 10contiguous nucleotides, more preferably at least 15 contiguousnucleotides, of a DNA molecule having a sequence as disclosed herein.Techniques for both PCR based assays and hybridization assays are wellknown in the art (see, for example, Mullis et al., Cold Spring HarborSymp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology, StocktonPress, NY, 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

In another embodiment, the compositions described herein may be used asmarkers for the progression of IBD. In this embodiment, assays asdescribed above for the diagnosis of IBD may be performed over time, andthe change in the level of reactive polypeptide(s) or polynucleotide(s)evaluated. For example, the assays may be performed every 24-72 hoursfor a period of 6 months to 1 year, and thereafter performed as needed.In general, IBD is progressing in those patients in whom the level ofpolypeptide or polynucleotide detected increases over time. In contrast,IBD is not progressing when the level of reactive polypeptide orpolynucleotide either remains constant or decreases with time.

In a further embodiment, the compositions described herein may be usedto monitor the level of antibodies specific for and/or T cellresponsiveness to an IBD-associated bacterial protein as a measure ofIBD progression. In general, IBD is progressing in those patients inwhom the level of antibodies that bind to a polypeptide or encoded by apolynucleotide described herein, that are detected increases over time.In contrast, IBD is not progressing when the level of reactiveantibodies either remains constant or decreases with time.

Certain in vivo diagnostic assays may be performed directly on a lesionin the colon. One such assay involves contacting cells from a lesionwith a binding agent. The bound binding agent may then be detecteddirectly or indirectly via a reporter group. Such binding agents mayalso be used in histological applications. Alternatively, polynucleotideprobes may be used within such applications.

As noted above, to improve sensitivity, multiple bacterial proteins maybe assayed within a given sample. It will be apparent that bindingagents specific for different proteins, antibodies, or T cells specificthereto provided herein may be combined within a single assay. Further,multiple primers or probes may be used concurrently. The selection ofbacterial proteins may be based on routine experiments to determinecombinations that results in optimal sensitivity. In addition, oralternatively, assays for bacterial proteins, antibodies, or T cellsspecific thereto, provided herein may be combined with assays for otherknown bacterial antigens or genetic markers such as the NOD2 mutation.

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a bacterial protein. Such antibodiesor fragments may be provided attached to a support material, asdescribed above. One or more additional containers may enclose elements,such as reagents or buffers, to be used in the assay. Such kits mayalso, or alternatively, contain a detection reagent as described abovethat contains a reporter group suitable for direct or indirect detectionof antibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a bacterial protein in a biological sample. Such kits generallycomprise at least one oligonucleotide probe or primer, as describedabove, that hybridizes to a polynucleotide encoding a bacterial protein.Such an oligonucleotide may be used, for example, within a PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding abacterial protein.

In an alternative embodiment, a kit may be designed to detect the levelof antibodies specific for an IBD-associated bacterial protein in abiological sample.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Identification of IBD-Associated Bacterial Antigensfrom a Mouse Cecal Bacteria Genomic Random Shear Expression Library

A mouse cecal bacteria genomic random shear expression library wasconstructed by sonicating C3H/HeJ Bir mouse cecal bacteria genomic DNAto produce fragment sizes of approximately 0.1 to 5.0 kbp. 14 μg ofsonicated DNA was treated with DNA polymerase I, Klenow fragment, for 30minutes followed by Pfu polymerase for 30 minutes to produce blunt endedfragments. EcoRI adaptors were then ligated to the fragments and thenadaptors were phosphorylated with E. coli polynucleotide kinase.Fragments were next fractionated with a Sephacryl 5400 column andfinally ligated to a Lambda ZAP Express (Stratagene) vector. Ligatedvector was then packaged with Gigapack III Gold packaging extract(Stratagene) and the unamplified library was plated with host E. coliXL-1 Blue MRF′ cells on LB agarose plates at a concentration of 25,000plaque forming units (PFU) per plate for 15 plates. After incubation at42° C. for 4 hours, nitrocellulose filters, soaked in 10 mM IPTG, wereadded and the plates which were incubated at 37° C. over night. Filterswere removed and washed 3× with PBS containing 0.1% Tween 20 (PBST),blocked for 1 hour with 1% BSA in PBST, washed 3× with PBST and thenincubated overnight at 4° C. in serum, preadsorbed with E. coliproteins, from C3H/HeJ Bir mice with inflammatory bowel disease. Afterwashing 3× with PBST, filters were incubated in a goat anti-mouse IgG,IgA, IgM secondary antibody conjugated with alkaline phosphatase for 1hour at room temperature. Filters were finally washed 3× with PBST, 2×with alkaline phosphatase buffer and developed with BCIP/NBT. Positiveclones were purified using the same technique; phagemid was excised; andresulting plasmid DNA was sequenced and searched against the GenBankdatabases. Those sequences that showed some degree of similarity toknown sequences in the database are listed in Table 2. Those sequencesthat showed no significant similarity to known sequences in the databaseare shown in Table 3.

TABLE 2 BACTERIAL SEQUENCES THAT SHOWED SOME DEGREE OF SIMILARITY TOKNOWN SEQUENCES IN THE DATABASE cDNA PRO SEQ SEQ Clone Clone Insert IDID Name ID kbp Blastn Blastx 1 38 Cbir-1 76779 1.3 No match (73%)flagellin A protein/ (48%) FlaB [Butyrivibrio fibrisolvens] (AF026812) 239 Cbir-2 76780 0.4 No match (73%) 3-isopropylmalate dehydrogenaseVC2491- Vibrio cholerae (group O1 strain N16961) 3 40, Cbir-3 76959 0.8No match (53%) motility protein A- 41 Termotoga maritima (strain MSB8) 5Cbir-5 76961 1.2 No match (44%) methyl-accepting chemotaxis protein[Bacillus halodurans] (AP001520) 6 42 Cbir-6 76781 1.0 No match (56%)ribosomal protein L6 (BL10) 7 Cbir-8 76962 3.0 No match (60%)acetohydroxy acid synthase large chain- Brevibacterium flavum 10 Cbir-1276964 2.0 (82%) (58%) phosphoenolpyruvate Same as carboxykinase VC2738-Blastx Vibriocholerae (group O1 strain N16961) 12 Cbir-14 76965 1.7 Nomatch (49%) gap regulator [Staphylococcus aureus] (AJ133520) 13 Cbir-1576966 1.0 (82%) (72%) flagellin A protein/ Same as FlaB (ButyrivibrioBlastx fibrisolvens) (AF026812) 14 44 Cbir-16 76967 1.2 No match (58%)ribosomal protein L6 (BL10) 15 Cbir-18 76968 0.8 No match (69%)flagellin - Roseburia cecicola (65%) flagellin A protein/ (63%) FlaB[Butyrivibrio fibrisolvens] (AF026812) 16 Cbir-19 77530 1.6 No match(51%) ABC-type transport protein-Synechocystis sp. (strain PCC6803) 1745 Cbir-20 76969 2.0 No match (68%) FlaB [Butyrivibrio fibrisolvens](AF026812) 18 Cbir-23 76970 0.8 (80%) (84%) elongation factor-Tu Same as[Porphyromonas gingivalis] Blastx (AB035462) 23 47 Cbir-32 76974 65 bpNo match probably amidohydrolase Campylobacter jenjuni (strain NCTC11168) 24 Cbir-36 77074 1.6 (87%) (83%) elongation factor TU- Bacillus 1(Streptomyces subtilis ramocissimus) complete genome 26 48 Cbir-39 769750.6 No match (70%) Thermotoga maritima (strain MSB8) (55%) flagellin Aprotein/ (56%) FlaB [Butyrivibrio fibrisolvens] (AF026812) 27 Cbir-4077075 0.7 No match (54%) flagellin-Thermotoga maritima (strain MSB8)/(58%) flagellin (Bacillus halodurans) (AP001512) 29 49 Cbir-44 76977 1.0No match (45%) flagellin-Thermotoga maritima (strain MSB8)/ (57%)flagellin (Bacillus halodurans) (AP001512) 31 Cbir-46 77533 2.2 No match(AL512975) 32 Cbir-49 77534 0.9 No match (59%) flagellin-Roseburiacecicola (56%) flagellin A protein/ (58%) FlaB (Butyrivibriofibriosolvens) (AF026812) 35 Cbir-62 77536 0.6 No match hemolysinsecretion protein precursor-Helecobacter pylori (strain 26695)/methyl-accepting chemotaxis protein (Helecobacter pylori) 36 Cbir-7377538 2.0 No match elongation factor - TS 37 Cbir-78 77539 1.0 No match(62%) flagellin-Roseburia cecicola (M20983) (57%) flagellin A protein/(59%) FlaB (Butyrivibrio fibrisolvens) 51 CB1-T2 73261 150 No match 36%Streptomyces Arabinosidase Secreted 54 CB1-T5 73264 1050 No match 33%Arabidopsis ClpC protease 55 CB1-T7 73266 650 No match 54% S. cerevisiaeAcetyltransferase 56 CB1-T8 73267 350 No match 28% Bacillus subtilisUnknown 58 CB1-T10 73269 150 No match ? 40% Deinococcus ABC transporter59 CB1-T11 73270 1700 No match 52% Borrelia burgdorferi Fructose-6-p 1-phosphotransferase 60 CB1-T13 73272 250 No match 56% Vibrio/Clostridiumalcohol dehydrogenase 61 CB1-T14 73273 260 No match ? 53% Bacteroidessurface Ag BspA 62 CB1-T15 73274 1000 No match 61% Clostridium prolyltRNA synthetase 64 CB3-T1 75037 1.7 ? 88% 65% Bacillus subtilisstreptomyces oligopeptide transport ATP- bind pro OPPF 67 CB3-T4 75040?>600 No match 36% E. coli/Staph Hypothetical 14.8 kDa membrane protein70 CB3-T9 75044 300 No match 91% Helicobacter pylolri2,3,4,5-tetrahydropyridine- 2-carboxylaten- succinyltransferase 72CB3-T12 75046 1700 No match 52% Thermotoga/Archaeoglobus/ Halobacteriumconserved prot. 74 CB3-T14 75048 1300 No match 29% Streptomyces heatshock

TABLE 3 BACTERIAL SEQUENCES THAT SHOWED NO SIGNIFICANT SIMILARITY TOKNOWN SEQUENCES IN THE DATABASE CDNA PRO SEQ SEQ Clone Clone Insert IDID Name ID kbp Blastn Blastx 4 Cbir-4 76960 2.1 No match No match 8 43Cbir-9 76782 0.6 No match No match 9 Cbir-11 76963 1.5 No match No match11 Cbir-13 77529 1.5 No match No match 19 Cbir-24 76971 2.3 No match Nomatch 20 Cbir-26 77073 1.2 No match No match 21 46 Cbir-27 76972 0.5 Nomatch No match 22 Cbir-30 76973 0.7 No match No match 25 Cbir-37 775312.5 No match No match 28 Cbir-41 76976 1.2 No match No match 30 Cbir-4577532 1.2 No match No match 33 Cbir-50 77535 0.7 No match No match 34 50Cbir-61 77076 0.5 No match No match 52 CB1-T3 73262 600 No match Nomatch 53 CB1-T4 73263 250 No match No match 57 CB1-T9 73268 250 No matchNo match 63 CB1-T16 73275 400 No match No match 65 CB3-T2 75038 500 Nomatch No match 66 CB3-T3 75039 500 No match No match 68 CB3-T5 75041 400No match No match 69 CB3-T6 75042 600 No match No match 71 CB3-T10 75045?>500 No match No match 73 CB3-T13 75047 700 No match No match

Example 2 Cloning and Expression of Flagellin X

A novel flagellin was cloned by PCR amplification from total cecalbacterium genomic DNA obtained from C3H/HeJ Bir mice. Oligonucleotideprimers were developed with sequence from the amino terminus of cloneCbir-1 (SEQ ID NO:1) and with a consensus sequence derived from thecarboxy terminus of three flagellin sequences that are most closelyrelated the Cbir-1 sequence. A six histidine tag and a Nde Iendonuclease cleavage site were added to the amino primer for cloningand for purification of recombinant protein and a Hind III cloning sitewas added to the carboxy-terminal primer. The full-length Flagellin XcDNA sequence (SEQ ID NO:75) was expressed as a pET17b (Novagen,Madison, Wis.) construct and resulting recombinant protein was purifiedwith a Ni-NTA affinity column (Qiagen, Valencia, Calif.). The fulllength amino acid sequence of Flagellin X is represented in SEQ IDNO:79. Both Western blot analysis and ELISA assays demonstrated thatthis recombinant flagellin protein was reactive with antibody from micewith IBD.

Three truncated forms of Flagellin X were constructed and used inexpression studies. The following truncations were cloned using PCRprimers developed from the flagellin X sequence:

-   -   i. the amino terminal conserved end of the molecule (cDNA        sequence: SEQ ID NO:76, amino acid sequence: SEQ ID NO:80)    -   ii. the amino terminal conserved end plus the variable portion        of the molecule (cDNA sequence: SEQ ID NO:77, amino acid        sequence: SEQ ID NO:81)    -   iii. and the carboxy-terminal conserved portion (cDNA sequence:        SEQ ID NO:78, amino acid sequence: SEQ ID NO:82)

A six histidine tag and cloning sites were added to all constructs forcloning and expression with the pET17b vector. The constructs wereexpressed in E. coli and recombinant protein analyzed by Western blotand ELISA. Western blot and ELISA assays with amino terminal and carboxyterminal recombinant protein demonstrated that the greatest antibodyreactivity was to the conserved amino terminus. These recombinantproteins have utility in the diagnosis, therapy, and vaccine developmentfor IBD.

Example 3 Identification of the Full-Length Nucleotide and PolypeptideSequence of Helicobacter bilis Flagellin B

This example describes the identification of the full-length H. bilisflagellin B nucleotide and polypeptide sequence. The recombinant DNA andprotein sequences have utility, for example, in the diagnosis, therapy,and vaccine development for IBD.

Infection with Helicobacter spp, including H. bilis, has been reportedto cause IBD in immunodeficient mice. Thus Helicobacter spp and specificHelicobacter proteins are useful tools for investigatingmicrobial-induced IBD. Helicobacter spp, especially H. pylori, have alsobeen implicated in human IBD. As shown in Examples 1 and 2 herein,bacterial flagellin are believed to be associated with IBD in the mousemodel. Described herein are the nucleotide and polypeptide sequences forH. bilis flagellin B (FlaB). This sequence was amplified using standardtechniques from H. bilis total genomic DNA using oligonucleotide primersderived from the H. mustelae flagellin B sequence. The full-lengthnucleotide sequence (coding sequence) of the H. bilis flagellin B is setforth in SEQ ID NO:83. The amino acid sequence encoded by thisnucleotide is set forth in SEQ ID NO:84. This polypeptide showedhomology to H. mustelae and H. pylori flagellin B proteins. The aminoterminal conserved region of the H. bilis flagillin B protein includesamino acid residues 1 to 154 of SEQ ID NO:84, and the correspondingnucleotides that encode this region from SEQ ID NO:83. Thecarboxy-terminal conserved portion includes amino acid residues 365 to514 of SEQ ID NO:84, and the corresponding nucleotides that encode thisregion from SEQ ID NO:83. In summary, these sequences representattractive therapeutic and diagnostic targets for IBD.

Example 4 Identification of the Full-Length Nucleotide and ProteinSequence of CBir-1 Flagellin

This example describes the full-length sequencing of the Cbir-1flagellin clone (partial sequence set forth in SEQ ID NO:1). This clonewas originally obtained by serologic expression screening a mouse cecalbacterium library with serum from mice with IBD (see Example 1 and Table2). The recombinant DNA and protein sequences have utility, for example,in the diagnosis, therapy, and vaccine development for IBD.

The Cbir-1 clone is a partial bacterial flagellin sequence (amino endplus variable region) that was shown to be highly reactive with themouse serum used to screen the bacterial library. Recombinant proteinderived from this clone and a recombinant representing the aminoterminus of this cloned sequence were also highly reactive with diseasedmouse (IgG2a) and human serum and also potentially contain a T cellepitope. These data indicate that this protein is immunogenic in mouseand in human. Described herein are the full-length CBir-1 flagellinnucleotide (SEQ ID NO:85) and protein (SEQ ID NO:86) sequences. Thenucleotide sequence was obtained by standard PCR amplificationtechniques from total genomic mouse cecal bacterial DNA with a primerspecific for Clone CBir-1 sequence and a second primer derived from theconserved carboxy end of related flagellin sequences. Polypeptidealignments confirmed that the protein is related to other flagellinproteins. The amino terminal conserved region of the CBir-1 proteinincludes amino acid residues 1 to 147 of SEQ ID NO:86, and thecorresponding nucleotides that encode this region from SEQ ID NO:85. Theamino terminal conserved end plus the variable portion of the moleculeincludes amino acid residues 1 to 418 of SEQ ID NO:86 and thecorresponding nucleotides that encode this region from SEQ ID NO:85. Thecarboxy-terminal conserved portion includes amino acid residues 361 to493 of SEQ ID NO:86, and the corresponding nucleotides that encode thisregion from SEQ ID NO:85. Thus, in summary, these sequences representattractive therapeutic and diagnostic targets for IBD.

FIG. 1 shows a schematic of flagellin clones with percent similarity torelated flagellin B from the rumen anaerobe, Butyrivibrio fibrisolvens.(A) Structure of B. fibrisolvens flagellin A showing conserved amino andcarboxyl regions and the hyper-variable central domain. (B) Mapping ofthe predicted amino acid sequences from the flagellin expression clones(1-12) in relation to the B. fibrisolvens sequence. Percentage range(41-84%) indicates the similarity in NH₂-conserved sequence between theclone and B. fibrisolvens sequences. (C) Diagram of the full-lengthamino acid sequence of mouse cecal bacteria flagellins cBir-1 andFla^(X) indicating the similarity of the three domains with therespective B. fibrisolvens domains. (D, E) Schematics of recombinantflagellin proteins and fragments for cBir-1 (D) and Fla^(X) (E) thathave been expressed and purified in E. coli by 6-histidine tag affinityto NiNTA columns (Qiagen Corp.).

Example 5 Peptide Priming of T-Helper Lines

Generation of CD4⁺ T helper lines and identification of peptide epitopesderived from bacterial-specific antigens that are capable of beingrecognized by CD4⁺ T cells in the context of HLA class II molecules, iscarried out as follows:

Fifteen-mer peptides overlapping by 10 amino acids, derived from abacterial-specific antigen, are generated using standard procedures.Dendritic cells (DC) are derived from PBMC of a normal donor usingGM-CSF and IL-4 by standard protocols. CD4⁺ T cells are generated fromthe same donor as the DC using MACS beads (Miltenyi Biotec, Auburn,Calif.) and negative selection. DC are pulsed overnight with pools ofthe 15-mer peptides, with each peptide at a final concentration of 0.25μg/ml. Pulsed DC are washed and plated at 1×10⁴ cells/well of 96-wellV-bottom plates and purified CD4⁺ T cells are added at 1×10⁵/well.Cultures are supplemented with 60 ng/ml IL-6 and 10 ng/ml IL-12 andincubated at 37° C. Cultures are restimulated as above on a weekly basisusing DC generated and pulsed as above as antigen presenting cells,supplemented with 5 ng/ml IL-7 and 10 U/ml IL-2. Following 4 in vitrostimulation cycles, resulting CD4⁺ T cell lines (each line correspondingto one well) are tested for specific proliferation and cytokineproduction in response to the stimulating pools of peptide with anirrelevant pool of peptides used as a control.

Example 6 Generation of Bacterial-Specific CTL Lines Using In VitroWhole-Gene Priming

Using in vitro whole-gene priming with bacterial antigen-vacciniainfected DC (see, for example, Yee et al, The Journal of Immunology, 157(9):4079-86, 1996), human CTL lines are derived that specificallyrecognize autologous fibroblasts transduced with a specific bacterialantigen, as determined by interferon-γ ELISPOT analysis. Specifically,dendritic cells (DC) are differentiated from monocyte cultures derivedfrom PBMC of normal human donors by growing for five days in RPMI mediumcontaining 10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml humanIL-4. Following culture, DC are infected overnight with bacterialantigen-recombinant vaccinia virus at a multiplicity of infection(M.O.I) of five, and matured overnight by the addition of 3 μg/ml CD40ligand. Virus is then inactivated by UV irradiation. CD8+ T cells areisolated using a magnetic bead system, and priming cultures areinitiated using standard culture techniques. Cultures are restimulatedevery 7-10 days using autologous primary fibroblasts retrovirallytransduced with previously identified bacterial antigens. Following fourstimulation cycles, CD8+ T cell lines are identified that specificallyproduce interferon-γ when stimulated with bacterial antigen-transducedautologous fibroblasts. Using a panel of HLA-mismatched B-LCL linestransduced with a vector expressing a bacterial antigen, and measuringinterferon-γ production by the CTL lines in an ELISPOT assay, the HLArestriction of the CTL lines is determined.

Example 7 Generation and Characterization of Anti-Bacterial AntigenMonoclonal Antibodies

Mouse monoclonal antibodies are raised against E. coli derived bacterialantigen proteins as follows: Mice are immunized with Complete Freund'sAdjuvant (CFA) containing 50 μg recombinant bacterial protein, followedby a subsequent intraperitoneal boost with Incomplete Freund's Adjuvant(IFA) containing 10 μg recombinant protein. Three days prior to removalof the spleens, the mice are immunized intravenously with approximately50 μg of soluble recombinant protein. The spleen of a mouse with apositive titer to the bacterial antigen is removed, and a single-cellsuspension made and used for fusion to SP2/O myeloma cells to generate Bcell hybridomas. The supernatants from the hybrid clones are tested byELISA for specificity to recombinant bacterial protein, and epitopemapped using peptides that spanned the entire bacterial proteinsequence. The mAbs are also tested by flow cytometry for their abilityto detect bacterial protein on the surface of cells stably transfectedwith the cDNA encoding the bacterial protein.

Example 8 Synthesis of Polypeptides

Polypeptides are synthesized on a Perkin Elmer/Applied BiosystemsDivision 430A peptide synthesizer using FMOC chemistry with HPTU(O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence is attached to the amino terminus ofthe peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support is carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides are precipitated in cold methyl-t-butyl-ether.The peptide pellets are then dissolved in water containing 0.1%trifluoroacetic acid (TFA) and lyophilized prior to purification by C18reverse phase HPLC. A gradient of 0%-60% acetonitrile (containing 0.1%TFA) in water (containing 0.1% TFA) is used to elute the peptides.Following lyophilization of the pure fractions, the peptides arecharacterized using electrospray or other types of mass spectrometry andby amino acid analysis.

Example 9 Flagellin-Reactive Antibody in Serum from Colitic Mice and itsRelationship to Pathology

Serum antibody response to recombinant flagellins cBir-1 and Fla^(X) andfragments were determined by Western blot analysis. One hundredmicrograms of full length protein (FL) as well as the amino conservedregion (A) and the conserved carboxyl region (C) fragments weresubjected to SDS-PAGE separation, transferred to membrane and subjectedto serum from non-colitic (pool of 2) and colitic C3H/HeJBir (pool of 5)mice. A second experiment was performed using 20 ng recombinant proteinand serum from non-colitic FVB (pool of 5) and colitic Mrd1a^(−/−) (poolof 5) mice. Human samples were used in a third experiment. Random humanblood donor and a Crohn's patient with severe disease were run against50 ng of recombinant protein. The results of these Western Blot analysesare shown in FIG. 2 a. Mouse cecal bacteria antigens (CBA) was used as acontrol.

FIG. 2 b shows titration of serum anti-flagellin antibody againstrecombinant flagellins cBir-1 and FlaX with secondary antibodiesspecific for mouse IgG and IgG2a antibodies. Serum from colitic mice(pool of 5) versus non-colitic C3H/HeJBir mice (pool of 2) was used inthe upper panel and serum from colitic MDR mice (pool of 5) versusnon-colitic FVB (pool of 5) mice was used in the lower panel.

FIG. 2 c shows the correlation of colitis score with serum anti-Fla^(X)antibody at a dilution of 1:200 (r=+0.7585). Correlations were alsodetermined for mouse age versus colitis score (r=+0.3716), and mouse ageversus anti-flagellin OD 450 at a 1:200 dilution (r=+0.3253). Colitisscores were based on the following scale: No disease (0-2); mild disease(3-15); moderate disease (16-35); and severe disease (≧36).

Similar antibody reactivity to recombinant CBir1 and Fla-X were seen inhuman Crohn's disease patient sera. As above, anti-flagellin antibodiesmeasured using ELISA in a panel of human serum samples from about 150donors with well characterized human inflammatory bowel diseases,ulcerative colitis and Crohn's Disease, as well as healthy controlspatients (Inflammatory Bowel Disease Center, Cedars-Sinai MedicalCenter, Los Angles, Calif.). A significantly higher level of serumanti-flagellin antibody was seen in Crohn's disease patients than innormal controls, infection controls and ulcerative colitis patients. Asignificantly higher serum antibody response was seen in pANCA-positiveCrohn's disease patients than in pANCA-positive ulcerative colitispatients. These results confirm that the antigens disclosed herein areuseful for subdividing IBD patients into categories of disease and wouldprove useful in clinical diagnosis of IBD, especially Crohn's Disease,FIG. 2 d.

Example 10 Flagellin-Stimulated Human Monocyte-Derived MacrophagesRelease Interleukin 10 and Tumor-Necrosis-Factor

Human monocyte-derived macrophages from seven healthy donors werecultivated in vitro for five days and then stimulated with full-lengthFlagellin X (SEQ ID NO:79) at 6, 12 or 24 μg/ml. Release of cytokinesIL10 and TNF-α was detected in the culture supernatant by ELISA.Cytokine release following stimulation with Flagellin X wasdose-dependent, see FIG. 3.

Example 11 Flagellin Proteins in Combination with Cholera Toxin Adjuvantas a Vaccine for IBD

Bacterial flagellin protein, FlaX, was used in conjunction with a knownadjuvant, cholera toxin (CT) to prevent the on-set of severe colitis inmice that are genetically prone to the disease. MDR1a mutant mice(MDR1aKO mice) develop severe, chronic inflammation in the colon as theyadvance in age. 5-6 week old MDR1a knock out female mice from TaconicFarms were either left untreated or given 8 μg FlaX or 8 μg Mtb+10 μgcholera toxin (List Biologics) in a 200 μl volume of PBS by oral gavage.Mice were thus treated on day 1 and day 14 of the experiment. Mice weresacrificed 4 weeks after the last dose, and their large intestines(cecum/colon/rectum) were fixed in 10% neutral-buffered formalin(Sigma), sectioned onto slides, and stained with hematoxylin and eosin(H&E). The sections were examined in a blinded fashion and scored fordisease severity.

Scores ranged from 0 (for no changes seen anywhere) to 64 (most severeinflammation and epithelial changes seen through the entire largeintestine) arbitrary units. Typically, mild disease scores as 1-15,moderate disease as 16-35, and severe disease as >35. The mean score foruntreated mice (n=4) was 23.50 with a standard deviation of 17.37. Themean score for mice given control protein plus CT (n=5) was 25.40 with astandard deviation of 18.43. The mean score for mice given FlaX plus CT(n=5) was 6.80 with a standard deviation off 4.38. The mean scores ofthe mice treated with control protein vs treated with FlaX weresignificantly different (Mann-Whitney nonparametric test, p=0.0159).Thus treatment of MDR1aKO mice with FlaX plus CT resulted in less severedisease.

Example 12 Identification of the Full-Length Nucleotide and ProteinSequence of CBir-11 Flagellin

The full length cDNA sequence of Clone ID 76963, Cbir-11 flagellin clone(SEQ ID NO:9) was identified. The nucleotide sequence of SEQ ID NO:87 isthe determined cDNA sequence for clone CBir-11. SEQ ID NO:88 provides apredicted translated flagellin-like sequence protein sequence encoded bySEQ ID NO:87. A second predicted translated sequence encoded by SEQ IDNO:87 is provided in SEQ ID NO:89, which is a phosphoesterase-likeprotein sequence.

Example 13 Creation of CBir-11 Flagellin Responsive CD4⁺ T Cells

CD4⁺ T cells were isolated from mesenteric lymph node cells (MLN) fromcolitic mdr1a^(−/−), C3H/HeJBir, C3H/HeJ.IL-10^(−/−) by BD™ IMAGanti-mouse CD4 beads according to manufacturer's instructions (BDBiosciences Pharmingen, San Diego, Calif.). Briefly, MLN cells werelabeled with anti-CD4 beads ands then placed within the magnetic fieldof the BD magnet. The unlabeled cells in suspension were removed and thecells binding the beads were washed and used in the CD4+ T cell culture.More than 99% of the cells were CD4+ by FACS analysis.

T cell reactivity to Cbir1 was determined by stimulation assay asfollows, 1.1×10⁵ T cells were incubated in duplicate in the presence of4×10⁵ antigen-pulsed and irradiated APCs (prepared according to Cong etal., J. Exp. Med. 187:855-64, 1998). APCs were treated with 1 to 100μg/ml CBir1 for 18 hours. On day 3 of culture, 0.5 μCi [³H]-thymidinewas added and the cells harvested 16 hours post post. T cells respondedto Cbir1. Non-specific activation via TLR5 or TLR4 activation wasexcluded by the lack of stimulation by Cbir1 or Fla-X onovalbumin-specific proliferation of CD4⁺ T cells from DO11.10ovalbumin-specific TCR transgenic animals.

A CD4⁺ T cell line reactive to CBir1 was generated by biolating CD4⁺ Tcells from MLN cells from C3H/HeJBir mice as described above followed byculture with splenic antigen presenting cells (APC) that were pulsedwith CBir1 (100 mg/ml) overnight. The cells were restimulated every10-14 days. The T cell line was responsive to CBir1 but not to Fla-X ora variety of other microbial, food and epithelial antigens.

To determine if this T cell line could induce mucosal inflammation, itwas adoptively transferred into C3H/HeJ-scid/scid recipients andquantitative histopathologic scoring performed 8 weeks post-transfershowed that this CBir1-specific CD4⁺ T cell like induced colitis in allrecipients of an intensity that was similar to or greater than thatinduced by CBA-specific CD4⁺ T cell lines, where as none of therecipients given these anti-CD3-activated C3H/HeJBir CD4⁺ T cellsdeveloped disease.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1-72. (canceled)
 73. An isolated polypeptide comprising an amino acidsequence having at least 90% identity to the sequence set forth in SEQID NO:79.
 74. The isolated polypeptide of claim 73, wherein thepolypeptide comprises an amino acid sequence having at least 91%, 92%,93%, or 94% identity to the sequence set forth in SEQ ID NO:79.
 75. Theisolated polypeptide of claim 73, wherein the polypeptide comprises anamino acid sequence having at least 95% identity to the sequence setforth in SEQ ID NO:79.
 76. The isolated polypeptide of claim 73, whereinthe polypeptide comprises an amino acid sequence having at least 96%,97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO:79. 77.The isolated polypeptide of claim 73, wherein the polypeptide comprisesthe sequence set forth in SEQ ID NO:79.
 78. The isolated polypeptide ofclaim 73, wherein the polypeptide is immobilized on a solid support. 79.A fusion protein comprising a polypeptide according to claim
 73. 80. Acomposition comprising a physiologically acceptable carrier and apolypeptide according to claim
 73. 81. An isolated polypeptide fragmentcomprising at least about 25, 50, 100 or more contiguous amino acids ofthe sequence of SEQ ID NO:79, wherein the polypeptide fragment isimmunologically reactive with an antibody that binds to the full-lengthpolypeptide of SEQ ID NO:79.
 82. A fusion protein comprising apolypeptide fragment according to claim
 81. 83. A fusion proteinconsisting of a polypeptide fragment according to claim 81 and a sixhistidine (SEQ ID NO:90) tag.
 84. The polypeptide fragment of claim 81,wherein the polypeptide fragment comprises: (a) an amino acid sequencehaving at least 95% identity to the sequence set forth in SEQ ID NO:80,which corresponds to the amino terminal conserved region of the sequenceset forth in SEQ ID NO:79; or (b) an amino acid sequence having at least95% identity to the sequence set forth in SEQ ID NO:81, whichcorresponds to the amino terminal conserved region plus the variableregion of the sequence set forth in SEQ ID NO:79.
 85. A fusion proteincomprising a polypeptide fragment according to claim
 84. 86. Theisolated polypeptide fragment of claim 84, wherein the polypeptidefragment comprises the sequence set forth in SEQ ID NO:80.
 87. Theisolated polypeptide fragment of claim 84, wherein the polypeptidefragment comprises the sequence set forth in SEQ ID NO:81.
 88. A fusionprotein consisting of a polypeptide fragment according to claim 84 and asix histidine (SEQ ID NO 90) tag.
 89. A kit comprising: (a) at least onepolypeptide fragment of claim 81; and (b) a detection reagent comprisinga reporter group.
 90. The kit of claim 89, wherein said at least onepolypeptide fragment is immobilized on a solid support.
 91. The kit ofclaim 89, wherein the detection reagent comprises ananti-immunoglobulin, protein G, protein A, or a lectin.
 92. The kit ofclaim 89, wherein the reporter group is selected from the groupconsisting of radioactive groups, fluorescent groups, luminescentgroups, enzymes, biotin, and dyes.
 93. An isolated polynucleotidecomprising a sequence selected from the group consisting of: (a) thesequence provided in SEQ ID NO 75; (b) the complement of the sequenceprovided in SEQ ID NO 75; (c) sequences having at least 75% identity tothe sequence of SEQ ID NO 75; and (d) sequences having at least 90%identity to the sequence of SEQ ID NO
 75. 94. A method of detecting thepresence of inflammatory bowel disease in a mammal, comprising the stepsof: (a) contacting a biological sample obtained from a mammal comprisingantibodies with a polypeptide of claim 73; (b) detecting in the samplean amount of antibody that binds to the polypeptide; and (c) comparingthe amount of bound antibody to a predetermined cut-off value andtherefrom determining the presence of inflammatory bowel disease in themammal.
 95. A method of detecting the presence of inflammatory boweldisease in a mammal, comprising the steps of: (a) contacting abiological sample obtained from a mammal comprising polynucleotides withat least one oligonucleotide that is at least in part complementary to apolynucleotide of claim 93; (b) detecting in the sample an amount of apolynucleotide that hybridizes to said oligonucleotide; and (c)comparing the amount of said polynucleotide that hybridizes to saidoligonucleotide to a predetermined cut-off value, and therefromdetermining the presence of inflammatory bowel disease in a mammal. 96.A method of monitoring the progression of inflammatory bowel disease ina mammal, comprising the steps of: (a) contacting a biological sampleobtained from a mammal comprising antibodies with a polypeptide of claim73; (b) detecting in the sample an amount of antibody that binds to thepolypeptide; and (c) repeating steps (a) and (b) using a biologicalsample obtained from the mammal at a subsequent point in time; and (d)comparing the amount of bound antibody in step (b) to the amount ofbound antibody in step (c) and therefrom monitoring the progression ofinflammatory bowel disease in the mammal.
 97. A method of monitoring theprogression of inflammatory bowel disease in a mammal, comprising thesteps of: (a) contacting a biological sample obtained from a mammalcomprising polynucleotides with at least one oligonucleotide that is atleast in part complementary to a polynucleotide of claim 93; (b)detecting in the sample an amount of a polynucleotide that hybridizes tosaid oligonucleotide; (c) repeating steps (a) and (b) using a biologicalsample obtained from said mammal at a subsequent point in time; and (d)comparing the amount of said polynucleotide that hybridizes to saidoligonucleotide in step (b) to the amount of said polynucleotide thathybridizes to said oligonucleotide in step (c); and therefrom monitoringthe progression of inflammatory bowel disease in a mammal.